Let's Talk Publication 259 Molecular Biology aa. Itis the process of duplication of parental DNA priorto cell division. After the cell division both the daughter gets one ‘complete copy of the DNA whichis identical to its parent. In replication parental DNA strand get separated and both strands act asa template forthe synthesis fits complementary copy through akey enzyme DNA polymerase. In this type of replication in which, each daughter conserves half part ofits parental DNA which acts asa template and hralfpartisnew which is complementary to parental form by DNA polymerase called semiconservative replication. Three models for replication proposed that are (1) conservative replication, (2) dispersive replication, and (3) semiconservative replication. In conservative replication one daughter has original parental dsDNA the parental dsDNA is transferred to one daughter and another daughter has completely new dsDNA because according to this ‘model whole parental dsDNA act as a template andin dispersive replication daughter has some part ofa template and some part is new in same DNAstrand. {a}Semi Conservative replication | _(b) Dispersive eolcation | _(c) Conservative replication ‘orignal DNA 1 1 A + 4 + + Fist replication Second replication Fig. : Types of replication Meselson and Stahl experiment (1957) If. coli cells grown ina medium withthe sole nitrogen source (NHI) contained N", the “heavy” isotope of nitrogen Adensty of ONA isolated from N15 containing ells greater than that of normal N” containing a cell The mixture of DNAof Nand N* can be separated by cesium chloride density gradient (CsCl) centrifugation. This technique was the basic of Meselson nd Stahl experiment. Experiment Esher Col cells were grown in a medium containing solo heavy nitrogen (N"), thus all the nitrogen in their DNA was (N°) then cellular DNA isolated and subjected to centrifuged in @ CsCl density gradient. The band of N* appear at the Bottom of falcon (tube). After that the cells had been transferred to light nitrogen (N") containing medium, cellular DNA isolated after one generation from cell grown in N containing medium after that subjected to centrifuged in a CI density gradient and found to be equilibrated at a higher position in centrifugation falcon. The hybrid band (N° NN") appears in the mid of falcon. To eliminate the confusion that DNA replication is semi-conservative or not. . coll cells were again allowed to double in number in the N medium for one more generation, thus E. col cell grown in two‘Molecular Biology 260 generation within in N* containing medium after that subjected to centrifuged in a CsCl density gradient and equilibrated which show two type of bands, one a higher position in centrifugation falcon then NY -N witha density equal to N DNA and another one share the same position like hybrid (N'-N") DNA. The disappearance of N- N" band indicates that the replication in semi-conservative in nature, When the cell was allowed to grow in N" medium forsomemore generation the NN" duplexnever observes again. Generation 1 20min 40 min —> ‘ — ‘ ‘ ‘ Gravitational oe —- NB ew Nas NE { Hybrid Heaw DNA DNA NN! Light ONA, NN N32. Hybrid DNA Nin heats Fig. : The Meselson-Stahl experiment after replication 1.2. DNAstructure DNAs double-stranded helical structure. It is made up of four types of nucleotide. Nucleotides have deoxyribose ‘sugar, a phosphate and nitrogenous base. The four types of bases are adenine (A) guanine (G), Cytosine (C) and thymine (T). ONAis a polymer of these four nucleotides a phosphodiester bond link the adjacent nucleotide in DNA. One strand of DNA binds with other strands of DNA through Hydrogen bonding between complementary base pais. Directionality in DNA In eukaryotes the DNA is linear. Linear DNA has two types of ends 5" phosphate end and 3° on end. In dsDNA, the strands are antiparallel in orientation, Directionality has consequences in DNA synthesis because DNA polymerase synthesizes DNAin S'~3' direction, 1.3. ORI (Origin of Replication) DNA replication starts from a unique point called the origin of replication (Ori). This is proof by denaturation mapping experiment carried out by Ross Inman and colleagues on bacteriophage scientist proof that initiation of replication loop always starts at the unique point, whichis richin AT base pair known as ORI (origin of replication), 13-mers 9-mers a —__EEE Sana H— 245 bp ori a Fig. : DNA consists of three mers of 13 bP and five mers of 9bP.Let's Talk Put 261 Molecular Biology Enzymes of DNA re DNApolymerase DNA polymerase catalyzes the polymerization of deoxynucleotide triphosphate (dNTP) and synthesizing a new DNA strand on a template strand, DNA polymerase adds new nucleotide at 3' OH of existing nucleotide. Thus the addition of new nucleotide at3' end make the growth of chain in5'~3' direction. This S’-3" polymerization activity, Is present in all DNA polymerase. DNA polymerase also has some other function like 5’ ~ 3 exonuclease activity responsible for removal of nucleotide from polynucleotide strand and 3’ ~ 5’ exonuclease activity responsible for proofreading to check correct complementary nucleotide addition on a newly synthesized strand. All these three activities are not universalin all DNA polymerases. DNA polymerase also is known as “Replicas”. Structure of DNA polymerase with its mechanism of polymerization All DNA polymerase use a common catalytic mechanism to polymerize nucleotide, DNA polymerase structure resembles to the right hand of a human. Ithas three domain which resembles lke thumb, finger and palm. A catalytic active site is presented in the conserved motifs of the palm region. This catalytic active site is composed of 8 sheet. DNA polymerase binds with two divalent metal ion (Mg” and Zn”) in this region, The Mg” is helped in the binding, between aspartate residue and phosphate (PO, *) of incoming nucleotide. Mg” and Zn* stabilise the interaction as these ions form salt bridges. Fig. : Mechanism of polymerization DNA polymerase exists in open and closed conformation. These confirmations are due to change in conformation of 0 helix of finger domain. When DNA polymerase is not associated with incoming dNTP, polymerase present in open conformation. After reading the template DNA the DNA polymerase recruits its complementary nucleotide. This complementary binding between template DNA and incoming nucleotide allows the finger to close. This is called closed configuration of polymerase. ‘Actually "fingers" are also responsible for the accurate placing of a template at the active site inducing 90° band in a backbone of template just after the active site, thus only first template base expose after primer, Metal ion mg+2 binds to 30H and get separate the oxygen to hydrogen O toH by reducing their affinity and create 3'0-, thus it acts as ‘nucleophilic and attack on a phosphate of incoming dNTP. Metal ion Zn” make interaction with triphosphate of dNTP and it is responsible to neutralize negative charge of dNTP phosphates and stabilize pyrophosphate (8 and y‘Molecular Biology 262 Let's Talk Publication ‘tol onan (conc toe ve 5 | ar ; ee Gone ay ween ‘ens a retin fe oo: : a 5 whermactery Fig. : Rotation of O-helix phosphate). The newly form DNA when exit the enzyme binds with thumb region and thumb region play important role in possessive reaction, for accurate positioning of primer and active site and strong union between polymerase andits substrate, Incoming eoxyribonuceoside triphosphate thumb tempine primer sand e20in hel fingers" primer sand “pa Eee] =m] wy we catacay Fig. : Polymerization and editing ‘The domain palm interacts with the minor groove of arecently added base pair by hydrogen bonding. This interaction ‘occurs only ifthe correct nucleotides added. ifthe wrong nucleotide added then it causes a distortion of the helixand results in slow catalysis is reduced. The rate of phosphodiester bond formation significantly when the wrong ‘nucleotide is added. This wrong nucleotide move towards proofreading site and the incorrect nucleotide are removed by3'-5 exonuclease activity. The exonuclease activity is located in an independent domain with ts own catalytic site.Let's Talk Put 263 Molecular Biology Processivity is a tendency of DNA polymerase to remain associated with DNA template rather than dissociate and reassociate. High processivity of DNA polymerase makesit more reliable. The speed of DNA replication depends upon processivity. The f-clamp enhance the processivity of DNA polymerase Ill. The DNA polymerase Ill has less tendency to get dissociate from template DNA strand due to fi-clamp. (clamp is a clamp-like structure, which allows the binding between DNA template and DNA polymerase 1.4.1.1. Fidelity (reliability/ accuracy) of ONA Replication DNA replications very accurate proves. However, the accuracy Is not 100%. Now LET'S TALK about the addition of one wrong nucleotide, Different event and how the accuracy increases due to different mechanism responsible for correct addition of nucleotide at the fidelity of DNA replication has three steps. 1. __Duetothe thermodynamics of base pairing one wrong nucleotide added per 10° 2. Base selection by polymerase one wrong nucleotide added per 10°. 3. Proofreading by polymerase one wrong nucleotide added per 10" Nucleotide selection DNA proofreading DNA polymerase anna ce and Dehra TTT) LI -Jittesndaugonarenaton P OMer anes Teteractee lathe taro Irenovd 5 onan ‘© wewona fl | ometines when proofreading Distortion is recognized by mismatch Tha ‘mechanism falsthe incorrect base repair enzymes and they remove repair causes mutation. Isinserted in the newly synthesizes ‘wrong base [ONA, This causes distortion of Hl eatkase Fig. : Mechanism of DNA polymerase 4. Post-replicative mismatch repair one wrong nucleotide added per 10" nucleotide. Mismatch repair last event to check and correct the addition of the wrong nucleotide and due to this event accuracy increase enormously, The difference in free energy between correct (G-C and A-T) base pairing and incorrect (G-T for example) predicts 1/100 mistakes. 1.4.1.2. The family of DNA polymerase There are seven different families of DNA polymerase A,B, C,D,X,¥, and RT. Family Examples Errorrate Function A Poll, 17, Taq 40% to10* Replication 8 Poll, RB69, Pola, 10to 10* Replication DNA polymerases Pola, 5,‘Molecular Biology 264 © Pollliasubunit. 101010 Replication D — Pold ios to 10% Replication X Pol, 2,p,0,TéT 10"to10* Repair, TLS Y —Din8, Umucd, 107 to 10" Mutagenic, TLS Translesin DNA synthesis. Dpo4, Dbh, Pol, n, 1.4.1.3. DNApolymerase in Prokaryotes a 2) 8) ‘There is five DNA polymerase. Palm is conserved in all families but finger and thumb present as similar secondary structure elements from different sequences. DNA polymerase: twas isolated by Arthur Kornberg in 1956 from E.Coli. Ithas single subunit and encoded by the polA gene. Itis the first DNA polymerase tobe identified. Polymerase | has three activities. 5'-3' exonuclease activity 3/5’ exonucleaseand 5'-3' polymerase activities. When Klenow treated the pol | with proteases. Pol, lis cleaved by proteases such as subtilisin or trypsin it gives two fragments: A larger C- terminal or Klenow fragment (residues 324-928 and mw 68 KO), has both the polymerase and the 3-5” exonuclease activities N-terminal fragment (residues 1-323 and mw 35 KD) smaller one, has the 5'-3" exonuclease activity. Thus, Pol, | contain three active sites on a single polypeptide chain, It has 5 3! exonucleolytic activity coordinated with the synthetic/proofreading activity. Polymerization rate of Pol | is 10 to 20 nucleotides per second. Role of DNA polymerase in recombination, nick translation, excise RNA Primersand repair. Nick translation during DNA repair and in vitro is mediated by 5' - 3° exonuclease activity. Ths is also called as forwarding exonuclease activity. DNA polymerase lis able to start replication in vitro at anickin DNA. Nickis breakage of a phosphodiester bond. Nick produces two types of ends 5' phosphate and 3' OH ends. The enzyme extends the 3-OH end at a point where a phosphodiester bond has been broken in a double-stranded DNA and the new segment of DNA\s synthesized by moving the existing homologous strand in the duplex. Thus one strand of a duplex DNA can be degraded and change by resynthesis of new material also used to introduce radioactively labelled nucleotide into DNAinvitro 3 2 y DNA polymerase I Sp riick Pol “s a ‘The S'- 3! exonuclease remove the existing. nucleotide and add new nucleotide at 3' OM. This process is known as nick translation. Fig : DNA polymerase | activity DNApolymerase Il It has seven subunits and encoded by polB gene. DNA polymerase Il play role in DNA repair when replication fork progressis blocked due to damage in DNA. DNApolymerase II: thas nine subunits and encoded by pol C gene. itis a main replicating enzyme in E.coli with dimeric structure. DNA polymerase Ill core enzyme contains. catalytic core which contains alpha (a) subunit responsible for DNA polymerase activity, Epsilon (e) subunit responsible for 3'- 5° exonuclease proofreading activity and theta (8) subunit whichLet's Talk Put 265 Molecular Biology 14.14, 7 amploader Pol Ii (Holoenzyme) Fig. : Structure of holoenzyme stimulates 5'~3' activity of «-subunit. DNA polymerase Ill holoenayme contains a dimerization subunit tall () which link the two catalytic cores together, a processivity component (the B clamp) and core enzyme in its every monomeric unit. DNA polymerase Il holoenzymes also contain a clamp loader which remains associated with its one monomeric subunit. ‘Two copies of clamp present in Pol Ill, also known as a sliding clamp which is responsible for holding each catalytic cores on to their template strands. A clamp isa homodimer. b clamp is crucial for processivity. The y clamp loader is required to load b clamp on DNA strand at the replication fork and it is @ group of 5 proteins (68'xy) or hheteropentamericand function in an ATP dependent manner. Firstly y loader bindsto 3’ end of DNAand thenit open B sliding clamp to encircle DNAin ATP dependent manner. Polymerisation rate of Pol ll 1000 nucleotides per second, DNA polymerase IVand V: Pol IV has one subunit and encoded by dnaB and Pol V has three subunits and encoded by UmuD'2C or UmuC, both come from ¥ family of a polymerase. Both of them has low fidelity on undamaged templates and they are able to replicate through damaged DNA. Thus members of this family known as translesion synthesis (TLS) polymerases or error-prone polymerases because allowing replication to bypass certain types of damage. A family of polymerase are error-prone because 3'~5' exonuclease activity is absent. DNA polymerase in eukaryotes Eukaryotic cells have approximately 15 types of DNA polymerases. However only three three DNA polymerases a alpha, 6 delta, and € epsilon required for nuclear DNA replication. DNA polymerase y gamma require for mitochondrial DNA replication. Other DNA polymerase associated with repair and translesion synthesis (TLS). For example, DNA polymerase eta (Pol n) take partin the DNA repairby translesion DNA synthesis. DNA polymerase etais also known as XPV. A mutation and loss of this gene cause the Xeroderma Pigmentosum. Xeroderma pigmentosum ‘mutation in polymerase eta (h) causes loss of DNA repair, when the damage is caused by Ultra Violet light. When the epidermal cells of skin get exposed to Ultra Violet light blister appear on the skin. DNA polymerase Alpha (Pol a) In prokaryotes the DNA replication is initiated by RNA polymerase however in eukaryotes the DNA replication is sted by DNA polymerase a. It is responsible for the initiation of DNA replication at autonomously replicating sequence (ARS) both are leading and lagging strand of DNA. The Okazaki fragment required DNA polymerase again ‘and again, Polymerases alpha has four subunits in which two subunit function as primase so that primase from outside not require and other two subunit act as DNA polymerase. DNA polymerases a has DNA binding domain. Pol a after‘Molecular Biology 266 Let's Talk Publi 142 143 synthesizing primer of 10 bases, it also synthesized 20-30 nucleotide of DNA. The DNA synthesis by polymerase ais, known as IDNA. a get replace by leading polymerase & in lagging strand and poly e (epsilon) in leading strand, This, phenomenon of pol replacementis called pol switching. Proliferating Cell Nuclear Antigen (PCNA) Itis homotrimeric and initially recognized as an antigen which is expressed during the DNA synthesis phase or S phase of the cell cycle within the nuclei of cells thus called PCNA. PCNA act as a processivity factor or DNA clamp for DNA, polymerases in eukaryotes. Replication factor C (RFC), which is a heteropentameric (RFC 1-5) act as loader of PCNA. PCNA and RFC analogue of B sliding clamp and loader respectively. During DNA damage, PCNA is Involved in the RAD6-dependent DNA repair pathway and get ubiquitinated. PCNA along with & take part in repair, especially post- replication epai ac aS os x Fig. : Structure of PCNA’ Primase DNA polymerase can not initiate the DNA replication done. It require @3'0H to add deoxyribonucleotide (dNTPs). That means to "prime" a reaction of polymerization of a monomer, the DNA polymerase synthesizes a short segment (sequence) of RNA that work as a primer. Primase is RNA polymerase. Thus DNA polymerase themselves cannot initiate DNA synthesis de novo. Primase is encoded by the dnaG gene in prokaryotes. In leading strand, only one primer requires but each Okazaki fragments hasits own primer. In eukaryotes Pol aactas primase. Helicases Helicases are hexameric ring shape protein which is mechanically separate the two strands of double-stranded hucleie acid by translocation on one strand in ATP dependent manner. Helicase translocates on DNA strand in particular direction which is popularly known as a polarity of helicase means either move in 5° ~3'direction or 3'-5" direction. Helicase functions in a various event like DNA replication, recombination, repair, transcription termination, RNA splicingand RNA editing, Amechanism used by helicase to performits function known askineticselectivity. Super families of helicases Superfamily 6superfamilies ‘Superfamily 1 ‘SFIAsubfamily has 3-5 translocation polarity (example Rep and UvrD) ‘SFB subfamily has5'-3' translocation polarity (example Rec D) SF, thas DEAD-box RNAhelicases (example RecQ) SF, 3°5' translocation polarity (example papilloma virus E1) ‘SF, 5'-3' translocation polarity (example 9P4 helicase of Bacteriophage T7) SF, '5'-3' translocation polarily (example Rho) SF, They have AAA+ motif example MCM, RUuV A, RUVB, RuvC), Helicase loader Helicase loader transfers the helicase to replication origin, These helicase loaders break the ring of hexamer helicase and allow the DNA to pass. DnaC act as helicase loader in prokaryotic and Cdc6 act as helicase loader in the eukaryote. ‘These proteins load the helicase on DNA. In yeast, Cdc6 is a highly unstable protein and get degraded rapidly with a half-life of <5 minutes. Cdt 1 prevent its degradation, Cdt-1 is conserved from yeast to humans. Examples of orthologs of Catt. in 5. pombe is cdt1 (cde10-dependent transcript 1), in Drosophila melanogaster is ‘double-parked’ or Dup, in Xenopus|aevisis Cdtl. Licensing factor isa protein which provide a license for DNA replication of start (just ike driving license to drive a motor). The origin of replication is required to start replication once per cell cycle. The start ofLet's Talk Pul 267 Molecular Biology 14.4, 14s. replication is known as firing. This lead to the development of an idea that the licensing factor does exist in the cel. These factors are synthesised inthe cytoplasm and transported to the nucleus. ‘The CdC-6 and Ct-1 are synthesized in the G, stage of cell cycle. These proteins bind with origin recognition complex (ORC) and fored pre-replication complex. This allows the binding of MCM. In S phage the CDK-cyclin complex phosphorylates the CdC-6. The phosphorylated CDC-6 is transported out of the nucleus and degraded by Ubiquitin-proteasome pathway, Thus Cdt-1 and CD6-6 actas licensing factor Single-strand binding protein Helicase unwinds double-stranded DNA two single-stranded DNA and those single-stranded regions of DNA get prevented from annealing by binding of single-strand binding protein, (SSB) SSB because separated strand having tendency to get reassociate and form duplex thus allowing the DNA replication machinery to perform its function. ‘SSP presentin viruses to humans. in Escheria coli SB presentin the homo tetrameric form. Inhigh salt concentration tetrameric form SSB6S binds to approximately 65 nucleotides of DNA, in contrast, low salt concentration dimeric form (SS8)35 binds to 35 nt DNA. The $S8 binds in a cooperative manner means binding of one monomer facilitate the binding of the second monomer and further so no. In eukaryotes SSB present in a trimeric form known asheterotrimeric RPA (Replication Protein A). SSBs function as monomersin many phage and virus. 3 s 5 Helicase Helicase Tetrameric Binding of SSBP Dimeric Binding of SSBP 3 Fig, : Diagram showing binding of SSB protein to single stranded DNA to prevent re-annealing DNA ligase Once the RNA primer has been removed and replaced by DNA through Pol |, then with the help of DNA ligase phosphodiester bond is formed between the adjacent Okazaki fragments One Okazaki fragment has 3' ol end and another fragment of Okazaki has 5! phosphate end. These two ends are seal by DNA ligase. Nick remains in leading strand sealed by DNA ligase. DNA ligase also requires energy to make phosphodiester bond. NAD (nicotinamide ‘adenine dinucleotide) work as a cofactor and source of energy for DNA ligase in E.coli whereas ATP used by T4 DNA ligase. DNA ligasesare presentin both prokaryotes and eukaryotes. Both enzymes undertake a two-step reaction in first step DNA ligase interact with ATP and form an enzyme-AMP ‘complex. The AMP in the enzyme-AMP complex becomes attached to the 5! phosphate of the nick, and then a phosphodiester bondis formed with the 3"-OH terminus ofthe nick, releasing the en2yme and the AMP.‘Molecular Biology 268 Let's Talk Publi 146, o o=p—O— Ge o=p—O— Gh , Ox gee? r ase? x K 6 On ase DNAse RAD orATP ON sse3 by oC Base 3 6 Io I o=b-o 4 | } 1a ceechaian TON Te ‘Topoisomerases ‘Topoisomerase s able to introduced nick in a single strand and double strand by cleaving the phosphodiester bond in single strand or both the strand of double-strand DNA thus itis a type of nuclease. There are two main classes of topoisomerase. In Escheria coli four individual topoisomerase present and get classified in above mention, two classes. ‘Type! Topoisomerase: It is able to produce a break in one DNA strand and cause the linking number to increase by one. DNA binds with in cleft of enzyme thus get placed near active tyrosine, Active-site Tyr tyrosine attacks as a nucleophile and break a phosphodiester bond in one DNA strand, cleavingit and generating a covalent S'-phosphotyrosyl protein- DNA linkage and free 3/ OH cause the formation of open conformation of the enzyme, this result ina gap in cleaved strand and the ‘gap bridge by enzyme. After that uncut strand passes through the gap cause the formation of the closed conformation of enzyme, asa result, liberated 3'-OH of cleave strand attacks the 5'-phosphotyrosyl protein-DNA linkage to religate the cleaved DNA strand. Type | Topoisomerase did not use ATP and responsible for catenane/decatenated, relaxation of supercoiling and knotting/ unknotting. E.g. Type | Topoisomerase which relaxes negative supercolling in E.coli, Type IL Topoisomerase of E.coli. and Type | Topoisomerase of calf thymus which will relax negative and positive £ ‘Topoisomerase (type |) H. Rotation of Ligation and nicking 3 free3' end enzyme dissociation Fig. : Mechanism of Topoisomerase |Let's Talk Put 269 Molecular Biology 1s. ‘Type I divide into two subclasses; Type | which form covalent intermediate at 5" end of DNA with changing the linking. number by one integer and Type IB which form covalent intermediate at 3’ end of DNA. with changing the linking number by two integers. ‘Typell Topoisomerase: Itis dimeric orin some cases tetrameric, able to produce a break in two DNA strand and cause the linking number to increase by two. It requires energy released by ATP hydrolysis to facilitate conformational change in enzyme not for cleave and rejoin DNA and responsible for catenation/decatenated, relaxation/induction of supercoiling and knotting/ unknotting. The general mechanism features the passage of one intact duplex DNA segment through a transient double-strand break in another segment. The DNA segment enters and leaves the topoisomerase through gated cavities above and below the bound DNA, which are called the N gate and the Cgate, enter through N gate and release through C gate, Two tyrosine present in each active site subunit and each tyrosine form 5’-phosphotyrosyl protein- DNA linkage with one DNA strand with 3/-OH in other sites of cleave strand, two ATP require in over all process. The broken DNAs ligated, and the second DNA segment isreleased, E.g. Type Il Topoisomerase in E.coli known as DNA gyrase which is a tetramer of two Gyr A subunit and two Gyr subunits, can introduce negative supercoils and decrease Lk (Linking Number) through the utilization of ATP. DNA strand cutting and rejoining done by GyrA which is inhibited by nalidixic a quinolone antibiotic and its fluorinated derivatives norfloxacin and ciprofloxacin. ATP hydrolysis did by GyrB which isinhibited by novobiocin, DNAgyrase uses ininitiation of replication and transcription. Other examples include Type IV Topoisomerase of E.coli. Type I! topoisomerases of eukaryotes can relax both positive ‘and negative supercoils but cannot unwind DNA or introduce negative supercolls in DNA. In vertebrates Type, I! Topoisomerase has two isoform lla. and iB. In Archaea, DNA gyrase belongs to type IIA and topoisomerase Vi belongs t0IB family of Typelll Topoisomerase, Toposisomerase (type Il) °° " g 0 oF, \ OH Pe 7 one et fotation of OH Binding and PA ® tree3'end nicking Ligation and ‘enzyme dissociation Fig. : Mechanism of topoisomerase I! tanec ‘Approcess of DNA replication in prokaryotes ‘Arepliconis a unit of DNA replication which possesses the origin of replication. Ifthe replication fork proceeds in both the direction the replication are said to be bidirectional and when it praceeds in one direction itis called unidirectional replication, Replication proceeds generally bidirectionally. Replicon should posses complete molecule of DNA, In prokaryote only one replicon is present. Thus entire chromosome act as a single replicon but in eukaryote each chromosome contain multiple origins of replication in turn divided each chromosome in multiple replicons. ‘According to the replicon model, control of replication depends on two component: First, replicator define as entire set of cis-acting DNA sequence thats enough themselves to carries out DNA replication, in fact, origin of replication is the part of replicator, but in eukaryotes origin of replication act as replicator themselves because DNA sequence within origin of replication sufficient to direct replication. Second initiator, a protein that recognizes and binds to specific DNA elementin replicator to activate initiation of replication.‘Molecular Biology 270 Let's Talk Publication InProkaryotes DNA replication takes placesin three steps-Initiation, elongation and termination, Initiation In prokaryotes, eg. Escheria coli the ori C is a 245 bp long sequence. Ori C contains a number of a repeat of two ‘conserve or consensus sequence, frst 13 mer consensus sequence with three repeat and second9 mer sequence with five repeats, 13 mer which Is rich in AT base pair is present upstream to 9 mer. The GATC sequence present at ori Cis methylated at the adenine base, Dam en2yme methylates the 6th position of adenine base. Only fully methylated DNA can undergo DNA replication in pro karyotes, DNA-A-ATP bends with R1, R2 and R4 boxes of 9 mer sequence. ‘These boxes are comparatively conserved than the R3 that is 9bp box. The bending of DNA-A-ATP complex displaces the Fis protein present at this boxes. The Fis interacts with 1 HF protein which causes binding at 9 bp sequence. This bending allows loading of DNA-A-ATP complex to 13 bp sequences. The DNA C nowbinds at 13 bp sequences. DNACis, helicase loader. It recruits the DNA B (helicase) at 13 bp sequence. Initiator protein DNA A make a complex with ATP ‘and DNA A-ATP binds to 9 mer sequence repeats on 2 fully methylated chromosome. Which stimulates cooperative binding of DNAA, Oric (E.coli) Initiator binding sites BiB i 9 9 3 3 3 245 bp Dnaa, o) +ATP Initial Open complex. Complex 5 Dna8 4g) Drab Dnac _ Preprining, © naa, complex 3 Fig. : Initiation of DNA replication in prokaryotesLet's Talk Pul am Molecular Biology Gyrase or Type Il topoisomerase workin front of replication fork responsible to induce negative supercoiling by which it causes a removal of torsional strain (overwinding) in the DNA generate by helicase movement in 5’ ~3' direction. SB protein binds to single-stranded DNA created by helicase to preventit reassociation. The length of duplex DNA for initiation of replication is probably less than 60 bp. This complex of DNA-A, DNA-B and DNA-C is called as pre-priming. complex. Once a sufficient size of replication forks being made by DNA-B helicase it recruits the primase enzyme. The primase causes a release of DNA-C complex from the ori and bind to helicase. The helicase-primase complexis called as primosome. After RNA primer synthesis the beta (i) clamp loaded on DNA with the help of 7 clamp loader through the ATP hydrolysis. This result in the increase the affinity ofa clamp to core polymerase and the loading of core polymerase on DNA then t binds to other components of DNA polymerase Ill and facilitated dimerization. This results in assembly of DNA polymerase Ill holoenzymes on DNA. After binding of polymerase, this complex called replisome. As primer get synthesised, primase get replace by core polymerase on leading strand template, this replacement occurs every time for each Okazaki fragments, DNA polymerase of leading strand move continuously along the template but the DNA polymerase of lagging strand can not move continuously thus creating a loop in the DNA. The single-stranded template must extend for the length of at least one Okazaki fragment before the lagging polymerase finishes one fragment and is prepared to begin the next. DnaB needs to be present on lagging strand template and moving in the same direction in which the replicating forks moving. This attaches the primosome to the lagging strand template because itis needed for priming Okazaki fragment synthesis every time, Dnact a ok Onaa ona-c DNA-Abinds at or DNA‘C works helicase loader and recruit the helicase ator Dna nap (helicase) Dar N / Dna-c~ Dnac‘Molecular Biology mm Let's Talk Publication After Helicase activity SSBP binds sar with single strand DNA Jn Dimeric form Helicase (Dna-B) Dna-G (Primase) Helicase recruit primase when sufficient size of replication fork is formed. Primer Primase synthesizes the RNA PrimerLet's Talk Publication 273 Molecular Biology eclamp binds at 3' end of RNA primer with the help of -clamp loader B-clamp recruit DNA polymerase Il, in both leading and lagging strand. DNA polymerase Ill start synthesizing the DNA. But both the polymerases ‘moves in opposite direction consequently semi dicontinous type of DNA replication. Fig. : Initiation of DNA replication‘Molecular Biology 278 Let's Talk Publication Elongation Inelongation, DNA polymerase polymerises the DNA strand by the addition of dNTP through the same mechanism. In prokaryotes primer get removed by DNA polymerase |. DNA polymerase | bind at 3” OH of previous Okazaki fragment and cause the displacement of RNA base of a primer by it's S'-3” exonuclease activity. Polymerase | also add new dNTPs at the 3' on end of Okazaki fragment by it's S'-3’ polymerisation activity. The last phosphodiester bond is bond by DNA ligase. In case of both leading and lagging strand and then synthesis of leading and lagging strand complete, Termination ‘Termination of DNA replication in Escheria Col. is completed through the use of termination sequences and Tus protein, Termination takes place within 350 bp region when both replication fork approach each other at termination ‘sequence which is characterized by the presence of unidirectional Ter sites or Terminator sites (Ter A~Ter land Ter K) of ~23 bp. These Ter sites recognize by unidirectional Tus (terminus utilization substance). Ter site present in two B,>B,> 8, mutation in B1 effect more than 8, and 8,, a mutation in B, effect more than B, and mutation in B, least effect the initiation of replication. italso reveals that an origin can function effectively in the presence of functional Aalong with any of twoB, elements. Origin recognition complex OR-C) is composed of six proteins act as an initiator and binds to AT-rich Aand B1with ATP dependent manner, ORC2-5 binds strongly but ORC-6 binds weakly. ORC-6 has a nuclear localization sequence. In G1. stage the ORC-6 is phosphorylated by CDK-2 and cyclin A. This phosphorylation causes transport of ORC-6 to the nucleus and it binds with remaining ORC-2-5. After that transcription factor ABF | (ARS binding factor) binds to the B3 element. This directs initiation by affecting chromatin structure at the A and B1 elements. After binding ORC recruit helicase loader Cdc6 with Ct1 protein Cdt1 enhance the activity of Cdc6. Cde6 recruit helicase known as MCM. The loading of MCM on ARS also facilitate by Cdt1 along with ORC’s ATP hydrolysis. The complex of ORC, Cat-1, Cdt-6 and MCMiscalledas pre-replicative complex(pre-RCs) Now in phase pre-replicative complex trigger in an active state by phosphorylation of Cde6 and Cdl through kinases name Cdk and Dak. Phosphorylation of Cdc6 leads to its degradation and not available for further initiation the replication. Now the MCM recruit CdC-45 and GINS, This complex is called as CMG (CdC-45, MCM-GINS) complex. CDt-45 and GIN-45 enhance the helicase activity of MCM and MCM causes unwinding of Ds DNA. After that polymerase, aget recruited by helicase and make RNA primer nucleotide. The polymerase a makes 10 nucleotide long. RNA sequence which works as a primer for DNA synthesis. A 20 base pair DNA also form by polymerase a called as initiator DNA, or “IDNA”. After forming the iDNA the polymerase a get dissociated from DNA template. Replication factor C(RFC),2 clamp loader then binds to the IDNA and loads PCNA (clamp) on DNA in ATP dependent manner with the same mechanism mention in prokaryotic replication, DNA polymerase efor leading strand and DNA polymerase 8 cn lagging strand also comes there with help of its clamp. A further mechanism similar to prokaryote rather than primer removal which carried out when leading strand and lagging strand synthesis get completed. This marks the transition from initiation to elongation stage.Let's Talk Publication 27 Molecular Biology POSSTSOWOSOWOSISTSIS OS ‘ARS. ARS. POOSRISISISDIGGIISIS. ‘onc. ‘onc. cide-6 cde-6 ‘ORC ORC cata | cata Cide-6 Cde-6; ORC ORC CCde-6 recruit the minichromosome maintenance protein (MCM) cat cat Cde-6 Cdc-6; mcm ORC cm mcm ORC vicm Phosphoryated CDC-6 is transported outside the nucleus ‘and degraded by Ubiquitin pathway proteosome cata po gett | ‘The CaK and Cdk kinase phosphoryate the CdC-6, mcm ORC cm J mcm ORC vicm‘Molecular Biology 278 Let's Talk Publi mcm mcm McM mcm gn gn Gn GN Polymerase-a. binds with template DNA and synthesize primer Pola Polka Polymerase-o first synthesize RNA and later its synthesize DNA. PrimerLet's Talk Publication 279 Molecular Biology iDNA (Dark) b Pol-o. IDNA (Dark) RNA Primer (light) (CD45 and GIN are not shown in below diagrams PCNA PCNA PCNA recruit polymerase-6 and polymerase-e Polymerase «tis replaced by polymerase & and polymerase «, This is known as polymerase switching, Polymerasec fj Polymerasee oe @. e | Lagging strand requires polymerase-ot again and again Polo. Pole‘Molecular Biology 280 Let's Talk Publication Se ns i ss | seemerterin FEN-1 : I—I-I-I-J=d x =b=b==9- 6— === ==. | ‘The gap is fill by polymerase-6 ‘O©GCOO6 6 99 9O®. 2 .9.9.9.9.9 Fig, : The DNA replication in eukaryotes. estates281 Molecular Biology 1.6.1, Primerremoval ‘The RNA primers are removed by an exonuclease (MF1) in yeast and the gaps are filed by the DNA polymerase 6 in yeast and nicks are sealed by DNA ligase in a gene. In human flap endonuclease 1 (FEN1) break the phosphodiester bond between primer and DNA, then RNase H removes the RNA primer. RNA primers of Okazaki fragments are ‘removed by exonuclease FEN |. DNA polymerase makes a complex with FEN 1 to direct nick translation as in E. coli DNA polymerase |. after that nicks are sealed by DNA ligase. DNA polymerase 6 responsible for displacing the S' end of the primer into a single-stranded RNA flap and cleavage of the short RNA flaps or for long flaps, get coated by single- stranded DNA binding protein replication protein A (RPA) and then sequential cleavage by Dna2 nuclease and FEN1. nick a A it ye gap riick rick | onsets ye ee DNA Ligases. 0-06-06 -0-0-0—. | DNA ligase End on” End gap “ eect Fig. : Showing the primer removal in eukaryotes.‘Molecular Biology 282 Let's Talk Publi 7. Chromosome telomere Telomeres Peirshd Speardh (heticetir ight) of DNA, felomeres Fig. : Telomere structure End replication problem In eukaryotes linear DNA is present thus eukaryotes face difficulty at the end of ONA replication because the first primeron each strands removed, there isno way tofill the gaps because no3'-the end is upstream direction and DNA polymerase cannot be extended in the 3’-95' direction. In prokaryote, there is no problem in filling all the gaps because 3"-end of another DNA is always upstream to serve as a primer as the DNA is circular in prokaryotes. DNA. ‘would grow shorter Ifnothing was done to overcome this problem, ‘Telomere protection in germ cell, stem cell, cancerous cell End of the eukaryotic chromosome known as telomeres. Telomeres possess a long array of multiple short tandem repeats of sixbases TTAGGG up to 20 to several 100 (vertebrates including humans), which are rich in G-T bases. The telomere sequence of Tetrahymena is TTGGGG. Telomerase possesses a small segment of RNA, complementary to the six-base-pair telomere repeat due to this region it recognizes the telomeres and reminds it what sequence toadd. The end replication problem is solved by telomerase enzyme. Telomerase isa ribonucleoprotein. Itis made up of RNA and protein. The protein has reverse transcriptase activity and RNA is complementary to telomere sequence present at the end of DNA. Telomerase enzymes a reverse transcriptase which is held by two protein p43 and p123 responsible for the catalytic activity of the enzyme thus called as TERT (telomerase reverse transcriptase). The human telomeric sequences TTAGGG, ‘The Grrich strand of a telomere is added at the 3'-ends of DNA strands by telomerase. The RNA component of telomerase or serves as a template for the addition of new DNA repeats at 3" end. The C-terminal part of the TERT protein contains the reverse transcriptase activity. RNA appears to be tethered to the N terminal part of TERT. This, allows the RNA to perform its template role by moving with respect to the active site of the enayme as each nucleotide is added to the growing telomere. When 3'-ends get elongated by telomerase after that the complementary strand can befilled by normal RNA priming followed by elongation by DNA polymerase D and joining by ligase. This process of protection available in a germ cell stem cell and cancerous cell because all of them possess telomerase enzyme.Let's Talk Pul 283 Molecular Biology The telomere with a G-rich repeated sequence. een Fig. : End-replication problem in eukaryotes Tana onal aan Te Raper tomer conpoenaytotresqunce FMA There is RNA : DNA hybridization. This pairing between Icon a prnars onto ata oF ON na ‘Nucleotides are added to the 3’end of the) G-rich strand the protein component of telomerase has reverse transcriptase activity. It synthesise RNA as template and nucleotides added to 3’ end of DNA. By this way the telomerase extends the parent strand of DNA A SPIO (afer several nucleotides have been — "Se the RNA template moves along the ONA, cI WVOW More nucleotides are added further, 5 SLEPIWOML Tirana el Ce later telomerase enzyme dissociates from telomere end. ‘Synthesis takes place on the complementary) strand filling in the gap due to the removal of the RNA primer at the end. Conclusion: Telomerase extends the DNA, filing in the gap due to the removal of the RNA primer, ‘The enzyme telomerase is responsible for the replication of chromosome ends. "=n Fig. : Replication of ends of DNA by telomerase enzyme.‘Molecular Biology 284 473 ‘Telomere repeat sequences have been conserved during evolution even though some variation is seen. Several monocotyledonous plants - TTAGGG, Aspergillus nidulans -TTAGGG, Aspergillus oryzae — TTAGGGTCAACA, Paramecium ~ TIGGGG, Arabidopsis ~ TTTAGGG, Many insects ~ TTAGG, Trypanosoma ~ TAGGG, Tetrahymena - ‘TIGGGG. Drosophila having an exception to this general pattern because in fruit fly telomeres consisting of tandem sequences generated by successive transposition of two retrotransposons (HeT-A and TART) instead of being synthesized by telomerase, ‘Telomere protection in somatic cell ‘Telomerase protection in a somatic cell is done with the help of a group of a protein complex known as Shelterin complex and protein in this group known as shelterin protein because those provide shelters to the telomere. In mammals, this protein is known as TRF1, TRF2, TIN2, POT1, TPA, and RAPI. Shelterin act ina specific way to remodel the telomere into a loop called a t-loop (for “telomere-loop") and in t-loop each protein protects telomere in a particular manner. TTAGGG repeat binding factor-1 (TRF1) binds to double-stranded telomere DNA with TTAGGG repeats, TRF2 paralog of TRF1 binds to the double-stranded parts of telomeres. Single-stranded telomeric DNA protected from endonucleases through binding of POT1 (protection of telomeres -1) to single-stranded 3'-talls of telomeres at just 2 nucleotides away from the 5'-end of the other strang, in turn, double-stranded telomeric DNA at S’end of other strand also get protected from 5'-end exonucleases. TPP1 is a POT1-binding protein and present as a partner of POT1 in a heterodimer. TIN? (TRFL-Interacting factor-2) plays an organizing role in shelterin complex, It connects TRF1 and TRF2 to TPP1/POT1 dimer. RAPI (repressor activator protein-1) interact with TRF2. Shelterin proteins specifically present onlyat telomeres throughout the cell cycle and nowhere else. 3 G-strand strand TRFL TRF2 Telomeric repeats Hoop Dloop Fig. : Telomere Protection in somatic cell ‘Telomere structure and telomere-binding proteins in lower eukaryotes Yeasts also have telomere-binding protein which protects the telomere ends by forming D-loop in which single strands donot get hide and those protein bear a resemblance to mammalian shelterin proteins like Taz1 binds double stranded telomere same as TF, Taz1 with the help of Rap! and Poz1 binds to @ dimer of Tpz1 and Pott same asTPP1- POT1 dimer and protect single-stranded telomeric DNA. All these protein bond the telomere DNA up to 180 degrees by protein: protein interactions proteins bound to the double-stranded telomere and also bound to its single- stranded tail285 Molecular Biology 174, Yeast Rap! binds directly to double-stranded DNA show evolutionary relationship to mammalian Rap1. Rif and Rif2 are two partners of RAPJin yeast. Single-stranded telomeric tall binds by other protein complex Cdc13, Stn, and Ten1..Ciliated protozoan Oxytricha in which telomere-binding proteins were firstly discovered havingname TEBPa and TEBPD, which are evolutionarily related to POT! and TPP1 in mammals binds with single-stranded 39-end of the organism's telomeres and protect them from degradation. In Schizosaccharomyces probe, Poti responsible for ‘maintaining their integrity instead of limiting the growth of telomeres as mammalian POT1 does. Indeed the loss of Poti can cause the loss of telomeres from this organism. Telomeric protection through G-quadruplexes ‘Telomeres are protein-DNA structures at the ends of eukaryotic chromosomes that protect chromosome ends from fusion and are vital in safeguarding genomic stability. In Tetrahymena tandem repeats of short G-rich sequences (TTGGG6) present at 3 strand of telomeres that project as a single-stranded DNA overhang and forming G-quarters, which is form in a cyclic array through synchronization or coordination of four guanine residue and multiple layers of this G-quarters leads to the formation of G-quadruplexes. This arrangement takes place through non-Watson-Crick pairing or Hoogsteen hydrogen-bonding with the help of centrally positioned cation. cs G2 External TTA loops StAL St Fig, : Telomere protection through G-quadruplex Diagonal loop Fig. : Stability of the telomere‘Molecular Biology 286 18, 19, 194. The Hayflick Limit ‘Anotmal human cellcan be grown in culture up to 50 generations (or cycles of subculturing) for a limited period after they enter period of senescence, in which they slow down and afterwards stop dividing, and lastly they attain a crisis stage and get die. This upper limit on the life span of normal cells is known as the Hayflick limit, discovered by Leonard Hayflick in the 1960s. But tumour cells do not follow any such limit. Turnour cell divides generation after generation, for an indefinite period because. Tumour cell contain telomerase enzyme which replicates the endof DNA. Cancer cell / immortal cell Number of cell Senescence 50 30 20 ° Time >. Fig. : Graph between number of cell division and time of normal cell and tumor cell Modes of Replication There are various mode of DNA replication - (1) Theta Replication, (2) Rolling Circle, (3) Linear Replication. Differ to each other with respect to the initiation and progression of replication, but all produce new DNA molecules by semiconservative replication. Rolling-circle replication It involves a single-stranded intermediate that seems to roll off the plasmid. Plasmid’s double-stranded DNA has positive and negative strand. In rolling circle replication double-stranded or that place within stem-loop structure get nicked by Rep A protein at the positive strand and free 3’ OH serve as a primer which get replicate by DNA pol Il in leading strand manner and displacing the 5’ end which isnon-template strand bya strand displacement reaction. The ends of the displaced strand are ligated to make a single-stranded circular DNA molecule. RNA polymerase makes a primer at the 5” end containing strand, thus also capable to act as a template for lagging strand synthesis, After synthesis of new strand, primer gets removed by DNA pol |. The ends of the displaced strand are ligated by ligase to makea second double-stranded plasmid. E.g. M13, © 174, F plasmid. F-Plasmid replication Most plasmids use one of two common mechanisms of replication, both of which depend on the host cell replicative machinery for replication (ONA polymerases, ligases, primases, helicases, etc. are usually supplied by the host cell).Let's Talk Publication 287 Molecular Biology (2) Rep A covalently (2) Rep A protein Binds at 0SO. attach it self to 5” and course Nick, generate 3'0H1 Phosphate of DNA. and 5/Phosphate Nicking and Covalent tachment rejoining by Repa to8’ phosphate — —_, () The ONA starts {@) DNA ligase remove the Rep Aand unwinding by helicas form phosphodiester bond between ‘S'P and 3'OH of newly synthesised DNA. ‘NA ligase RNA primer RNA Pol so sso { — + ( } Pol ll CCC DNA SSDNA CCC DNA. (7) Covalent closed circular DNA is formed.) (((6) Polymerase lll extend the primer) { (5) The RNA primer is required to replicate the 2" strand of DNA. Fig : Mechanism of roling circle replication oro 1.9.2. Thetareplication- Itisa normal mode of DNA replication which resembles the Greek letter theta (8) during replication. The theta mode ‘of DNA replication isbidirectional in nature. oVFoy: Replication fork Fig. : Mechanism of theta replication‘Molecular Biology 288 193 19.4, Linearreplication— ‘This the type of replication takes place in linear chromosome like in eukaryotes. Gf Ateach fork, the leading strand synthesis takes place continuously in the same direction as that of Replication fork. Leading Lagging y strand strand 3 $ 5 ——_\V¥ Lagging Leading ff —__, Direction of strand strand Direction of Unwinding +> Replication fork Origin f Meach fork, the lagging strand synthesis takes place continuously in the opposite direction as that of Replication fork Fig. : Mechanism of linear replication Replication of organelle DNA (Models for mtDNA replication) DNA polymerase ys used exclusively for mtDNA replication and proofreading. Two models have been proposed for the mode of mtDNA replication, 1) strand displacement model, appeal to continuous DNA replication and 2) coupled ‘model, proposes semidiscontinous DNA replication. Strand displacement mode! ‘The strand displacement model (also called the strand asynchronous model) for mammalian mtDNA replication is the most widely accepted, longest standing model. In strand displacement model, replications unidirectional around the circle and there is one replication fork for each strand in which One strand is called the heavy strand (H) and another strand is called light strand (L). The designation of the strands as H and L due to their different buoyant densities in denaturing CsCl density gradients centrifugation. Two origins (OH and OL) because here one priming event per template strand and it starts within a region called the displacement or “D" loop of 500-600 nucleotides. The RNA primer synthesis by mitochondrial RNA polymerase. Firstly replication starts on H strand starting at OH. When DNA polymerase polymerise approximately two-thirds of the mtDNA as a result replication fork get passes the major origin of L strand synthesis, thus exposing the site ina single-stranded format which new H strand synthesis starts from OL. ‘Synthesis is continuous around the circle on both strands. Multifunction endoribonuclease, RNase MRP cleave RNA primer. The same mechanism for cpDNA (cytoplasmic DNA) replication. Strand coupled model In strand coupled model at multiple sites lagging strand replication (L strand) get initiated, showing a discontinuous synthesis of short Okazaki fragments thus requiring multiple primers. So, inthis model the coupled lagging strand and leading (H strand) synthesis represents a semidiscontinous, bidirectional mode of ONA replication. The endoribonuclease, RNase MRP (mitochondrial RNA processing) has at least two functions, 1) removal of RNA primers in mtDNA replication, 2) pre-ribosomal RNA's nucleolar processing. Mutation in RNase MRP cause cartilage-hair hypoplasia a rare form of dwarfism and due to its consequences multiple phenotypic manifestations (pleiotropy) occur which included short limbs, short stature, fine sparse hair, impaired cellular immunity, anaemia, and predisposition to several cancers.Let's Talk Publication 289 Molecular Biology 24. 24a. 21.2 DNA repair is the processes which maintain the integrity of the genome by correcting any kind of mismatch which takes place in DNA as well as repair in any kind of DNA damage occur by any means. ifa repair fails then a mutation occurs, There is various kind or repair system involved in different repair pathway depends upon the type of mismatch and DNA damages. ‘Type of DNArepairandtheirmechanism Mismatch repair (MMR): ‘This repair has important function during ONA replication because repair each mismatch precisely occur during, replication, here mismatch occur specifically in new form daughter strand and this strand easily recognize from parent strand because present in hemimethylated form and parent strand which is present fully methylated form, as well as this system, also recognize the presence of inserted or deleted base in DNA strand and its complementary strand Which s loop out or bulge out due to this insertion or deletion. One of the prominent cause of mismatch to occur is base flipping, which isa natural process occurs in dsDNA, in this process base flip for 10-8 secto its original position to opposite site and after 10-8 sec back toits original position. One another cause of mismatch to occur is replication slippage, it occurs if replication proofreading mechanism gets fall Firstly mismatch base pair gets recognize after that mismatch base gets scrutinize from the mismatch base pair which leads to excision of mismatch base and addition of correct one in its place by the function of much gene product, helicase, ligase, and polymerase. Mismatch repairin prokaryotes In prokaryotes methylation on DNA done by DNA adenine methylase (Dam), Dam cause methylation of Adenine at 6th position, which present in S'GATC3’ sequence located specifically on oriC as well as throughout the bacterial genome and mismatch repair is easily perform if mismatch occur within >1 kb region from GATC site, here this is repair with respect to E.coli. DNA methylation occurs after the DNA synthesis and the time duration between these two processes allow the mismatch repair to perform their function, in turn, recognize mismatch containing hemimethylated daughter strand. In E.coli various type of repair system involve depending upon the length of unmethylated strand name as the short patch, long patch, and very short patch. Here we mention repair of @ long. patch: Firstly recognition of mismatch base pair containing hemimethylated daughter strand carried out by MutS and MutS bind to mismatch as dimer and after that Mut, bind to MutS as dimer, binding of both of them leads to translocation of ‘Mut$ on DNA strand in ATP hydrolysis-dependent manner until it encounters GATC sequence, as it encounters GATC sequence, in turn forming a loop in DNA. After that Mutli (endonuclease) join the MutS-MutL complex at GATC sequence by recognition of hemnimethylated GATC sequence and Mut H cleave the unmethylated strand at GATC, this leads to excision of tis stand up to the mismatch site by the action of exonuclease, which occur in either direction, in 3't05' direction by the action of exonuclease | and in 5'to3* direction by the action of exonuclease Vil or Recl. After ‘that DNA get synthesis by DNA polymerase Mismatchin eukaryotic Mechanism of mismatch is same in both prokaryotes and eukaryotes, in eukaryotes homolog of but genes are as follows: MSH isa homolog of Mut. MLH isa homolog of Mutl MHH isahomolog of Muth Here Polymerase 6 responsible for the new DNA synthesis. In human, HNPCC (hereditary non polyposis colorectal cancer), a cancer disease occur due to mutation in hMSH and AMLH, it occurs due to microsatellite instability, ‘microsatellite instability is rapid change in the length (or repeats) of microsatellite sequence which occur due to replication slippage and this slip not get corrected by the MMR, due to mutation in MMR mediating genes,Molecular Biology 230 Let's Talk Publication 22 oe Methyl group — Mutt, Muts, Muti (mismatch repair proteins) (0) Nick Muth Methyl group Methyl group omar mans Methyl group Fig. : Mismatch repair Excision repair Excision repair work on DNA damage site, firstly remove the damage bases then resynthesis normal bases on the place of damaged bases. Excision repair also removes modified or incorrect base which forms by various mean and halts the function of anormalbasein modified form. Excision repair falls intotwo categories: Base excision repair (BER): ‘This type of repair function when the base gets modified by any mean and this modification halt the function of a normal base, thus for a proper functioning of genome removal of that base is necessary. Base gets modified by alkylating agent, deaminating agent and oxidative agent. Firstly modified base get recognize by DNA glycosylase enzyme and that enzyme removes get diffused into the minor groove of DNA and cleave the N-glycosidic bond the modified base, in turn, remove the modified base, as a result,Let's Talk Publication 291 Molecular Biology Benerate abasicsite or AP site (apurinicor apyrimidinicsite). In some cases modified base get to flip out from the helix and due to this flip base get a place into the active site of an enzyme incorrect location and this base gets remove as wellin DNA damage case, this base immediately gets excised in turn create AP site, As AP site get form to recognize by AP endonuclease, in turn cause the cleavage at site of polynucleotide strand, as a resulta segment of DNA\ncluding AP site is removed, after that incase of prokaryotes DNA polymerase lis synthesis ‘the damaged strand and ligase seal the nic, in case of eukaryotic polymerase 6/e get recruited which synthesis 2t020 nucleotide with FEN | enzyme along with DNA ligase 1 which seal the nick for long patch and for short patch APE | recruit polymerase B which replace singlenucleotide along with ligase 3 or XRCCI which seal thenick. Seay NA elvcosylase (b) ap ‘endonuclease ) 5 toy H 35 DNA polymerase NTPs Jeoxyribose phosphate + dNMPs cl) “SOT te) SE LEEL ddbddede: Fig. : Base Excision RepairMolecular Biology 292 Let's Talk Publication A 8) 23. ‘AP endonucleasetwo types: Monofunctional, which only actas endonucleases. Example- AGG(alkyl adenine glycosylase) act on hypoxanthine, 3- methyladenine, and 7-methylguanine. Uracil DNA glycosylase, one in bacteria but four in human and most common is UNG, remove U during ONA replication, hsMUGI remove U from single-stranded "DNA during replication and transcription. Due to deamination of S methylcytosine or cytosine generation of Tand U takes place respectively and both base pair with G, both U and remove by TSD remove MBD4. Bifunctional, which act as endonuclease as well a glycosylases. Example- MAG act on methyl guanine and ogg-lact onoxyguanine. ‘AP endonuclease carried out two types of cleavage Belimination: remove sugarat AP site lke that the generation of 3'0H and5'P takesplace. 65 elimination: remove sugar at AP site lke that the generation of S'OHand3'P takes place. Nucleotide Excision Repair (NER) Inthis repair mechanism, stretch of DNA or oligonucleotide get removed which include the damaged base, this done by cleaving the phosphodiester bond on each side of the lesion. This repair mechanism also carried out when DNA damage was done by UN. Inprokaryotes In prokaryote, uvr gene products called ABC exonuclease, which having three subunits, mediate the repair process alongwith the help of polymerase, helicase, and ligase. Here we mention the case of thymidine dimer, Firstly UvrA recognize the dimer or damage site and get bind toit, after ‘that UvrB comes and Uvr8 bind to UvrA at the 3'ste of the lesion, this leads to binding of UvrC at the S‘site of the lesion and removal of UvrAin ATP hydrolysis-dependent manner, because removal of UvrA facilitate binding of UvrC. Both UvrB and UvrC had endonuclease activity and mediate the cleavage on both sides of the lesion, UvrB cleave 3to 4 nucleotide at 3'site from the lesion and Uvr8 cleave 7 to 8 nucleotide at S‘site from the lesion, in turn, release 11 to 412 nt starch of DNA or we can say UvrBC dimer mediate the excision of oligonucleotide, as a result, recruitment of helicase Il or UvrD along with DNA polymerase | and ligase occur helicase remove the strand, DNA polymerase synthesis the new strand and ligase seal the nick During transcription, damage cause halt of transcription then Mfd protein comes at that site and displace the RNA polymerase and recruit the Uvr complex to carry our repair pathway, then next polymerase able to carry out transcription. TISDIS0 a we | = | DNA ligase, Pol. | Fig. : Nucleotide Excision Repair in ProkaryotesLet's Talk Publication 293 ‘Molecular Biology 232 23.24 Ineukaryotes: In eukaryote two types of nucleotide, excision repairs occur: first- transcription couple NER, this repair mechanism active when damage occurs in front of RNA polymerase during transcription or we can say damage occur in active {gene and second- global genomic NER, this repair mechanism is active when damage occur anywhere in the genome. After some initial step both mechanisms follow the same step for the completion of repair, so here we mention the initial step of both pathway separately then the common step we explain together. ‘Transcription couple NER: ‘The RNA polymerase Il recognize the UV damage lesion, in turn, degradation of the large subunit of polymerase takes place, TeFilH also presents here. Firstly two protein CSB and CSA bind to the damage lesion, then the XPG/Rad 23 (also ‘get bind by RPA) cause unwinding of DNA up to ~20 bp around the lesion and it requires the XPG govern ATPase activity because ATP hydrolysis requires for helicase movement and both proteinsinclude within the TeFIIH. After that XPG comes and binds to CSA/CSB at §’ site of lesion or in front of RNA polymerase, its binding leads to the binding of XPF to XPG at 3’ site of the lesion, both XPF and XPG had endonuclease activity. Further process same to global ‘genomicrepair. mRNA DNA damaging agent DNA templateMolecular Biology 294 Let's Talk Publication Fig. : Transcription couple NER 2.3.2.2, Global Genomic Repair: Here lesion get recognize by XPC, lesion occur anywhere in genome but if this lesion is CPD (cyclobutane pyrimidine dimer) then it gets recognize firstly by DNA damage binding protein (0DB1/2}, After binding of XPC or DDB to lesion binding of XPA with XPC or DDB occur, after that XPD binds to XPA and XPB binds to XPD and both protein perform their function which is same as mention in transcription couple NER. After that XPG comes and binds to XPC at 5" site of lesion or in frontof RNA polymerase, its binding leads to the binding of XPF toXPB at 3’ site ofthe lesion, both XPF and XPG had endonuclease activity. Here RPA binds to both XPC and XP G. Cleavage on either side of the lesion mediates by XPF and XPG. XPF cleave 6 nucleotides (nt) at 3’ site from the lesion and XPG cleaves 22 nucleotides (nt) at 5’ site from the lesion, in turn, release 28nt DNAstarch, as result recruitment of DNA polymerase 6/e for DNA synthesis and XRCCI for nicksealing takes place.Let's Talk Publication 295 Molecular Biology Soom rt pllililissliliils a, are a c STOTT TTT x AA » : Wilé-type ONA Opening af ONA double hel NA polmerase DNA igase xP-Fandxp6 3 endonucleases Fig. : Global genomic NER XP genes are the complex of eight (KPA to XPG), which mediate this repair mechanism. Mutation in XP gene leads to Xeroderma Pigmentosum, an autosomal recessive disease, in which a patient had hypersensitivity towards sunlight and specifically UW light. Cockayne syndrame result from mutation occurs in XPD /XPB (helicase mutation) or cSA/CsB, 2.4. SOSresponse ‘When DNA get damage severely then it requires immediate excision repair mechanism which carried out by the product of SOS responsive gene, whose product also important for cel survival because play important role in repair, recombination, cel division, mutagenesis, and replication. Activation of RecA protein occur by the signal generated due to DNA damage, in turn, replication halt and that activated RecA cause cleavage of LexA repressor at Ala-Gly peptide bond to inactivate LexA, because LexA is a repressor of SOS responsive gene and by bind to SOS box (20.bp) at SOS responsive gene promoter cause repression of ‘these genes, as LexA inactivate, activation of SOS responsive gene takes place and further repair process takes place. ‘This whole process collectively called SOS response. Cell distress signal-DNA damage Normal, no) Induces RecA protease function DNA damage ws ‘SOS DNA repair genes repressed by LexA protein are feed for — S expression loa eit feca Mactivates xAlproteln LexA protein Error-free DNA repair Error-prone DNA repair Repressed SOS operon ‘Active SOS operon 88080" Fig, :$OS responseMolecular Biology 296 Let's Talk Publication 25, In prokaryotes due to severe DNA damage, DNAstrand cannot be replicated by DNA polymerase il and then t recruit the DNA polymerase V (UmuD'2C). Cleavage of UmuD dane by RecA during severe DNA damage and production of Umub’ occur when two UmuD’ subunit combines with UmuC form (polymerase V) Umub’2C. When polymerase V bind to the lesion then add one correct nucleotide but not able to read the second nucleotide thus add incorrect one and after that release, which leads to recruitment of DNA polymerase Illand further replication continues. In Eukaryotes, during this type of damage DNA polymerase 8/ replace by DNA polymerase n, which add on it first nucleotide of dimer correct then release after that recruitment any of (i/t/w/rev-1/k) one polymerase takes place which add the wrong nucleotide and after that release, which leads to recruitment of DNA polymerase Ill and further replication continue. Dueto the addition of the wrong nucleotide called error-prone repair or translesion repair. Direct repair: DNA damage can directly be reversed by changing the damaged base into normal or damage base simply remove and replace by a normal one, this type of damage repair known as direct repair. One of these repair systems is known as photoreactivation (a non mutagenic repair system), in this repair pyrimidine dimer get repair by a light-dependent enzyme that binds to dimer and cause reversal of inappropriate covalent bond between adjacent pyrimidine residue ‘and convert into monomeric pyrimidine containing nucleotide. In .coli phr gene product name DNA photolyase, an enzyme. DNA photolyase gets stimulated by a wavelength of 300 to 500 nm (biue light) and at this wavelength, DNA photolyase binds to pyrimidine dimer and cause reversal of inappropriate covalent bond between adjacent pyrimidine residue and convert into monomeric pyrimidine containing nucleotide. This repair is not universal but widespread, specifically present in bacteria and plant. Interstrand pyrimidine dimer able to take the form of 6,4-photoproduct or CPD (cyclobutane pyrimidine dimer), but 6,4 photoproduct more mutagenic then CPD. The least common form of pyrimidine dimer is cytosine-cytosine dimer and most commonisa thymine-thymine dimer. Conversion of one type of base into other results in distortion of DNA structure and itis due to replication error, for example, conversion of cytosine to uracil by deamination, caused by chemical mutagen form mismatch and this, mismatch visualize in a future generation. Alkylating agent converts guanine into 0'6-methylguanine, which is a highly mutagenic lesion, in turn, form mismatch because now 0'6-methylguanine pair with thymine in spite of lo + ls axxetogutarate N ath: = on y ly (Ly j 2 man Ad emaaene § LY Il l I sec ie ‘ | eesanacieem cytosine -Methyleytosine Fig. : Direct DNA repairLet's Talk Publication 297 Molecular Biology 26. 27. cytosine. An alkyl transferase called 0'6-methylguanine DNA methyltransferase mediates the transfer of methyl group of 0'6-methylguanine to a specific cytosine residue, which is present within same protein, asa result, repair of 0’6-methylguanine into guanine takes place. Recombination repair: ‘This repair also known as post-replication repair because gap remain in daughter strand due to the presence of dimer in the parent strand, at this situation, replication cannot be stopped, thus in this condition we need to bypass the damage and synthesis the gap containing daughter strand and that gap in daughter strand fill by the help of normal duplex by taken it single strand or mediate single strand exchange, which acts as template for gap filing. The gap creates innormal strand get filly the repair process, in turn, form the normal duplexstrand, Replication fork stalls due to two reason, first if the lesion present in template strand and second nick present in parent strand, restoration of the fork in both case done bya separate method which explains below: If replication fork get stalls due to damage in the parent strand at the damage site, at that situation, stall fork (look like Holiday junction) get revert up to short distance that allow the pairing between two daughter strand and the first daughter strand get form by DNA polymerase by the acting of second daughter strand act as template for the first daughter strand synthesis because we know one daughter strand has long than other in this case and after the synthesis of the daughter strand to which lesion containing parent strand act as template, After that restoration of fork done by forwarding branch migration of homologous recombination, which is done by the action of helicase, Some protein name RecA, RecF, RecR, and RecO help in this process. If replication fork stall due to the presence of nick in one parent strand, in turn, double-strand break present in one double strand daughter, as a result, replication fork collapse occurs (lost of replication fork). Here homologous recombination carried out to fill the breakin both duplexes by the help of RecBCD, RecA, and RuvABC. In this process free3’ end get generated by RecBC0, then it become able for invasion by the binding of RecA oni, in turn cause strand invasion of free 3" into donor duplex and forming D loop which is homologous and act as template for DNA synthesis by DNA polymerase, as a result formation of Holiday junction takes place and branch migration done by RuvAB then Holiday junction resolve by RuvC and the restoration of replication fork done by cleavage of the Holiday junction. Repair of Double-strand break: Double strand DNA break get arise due to replication error, ionizing radiation and the oxidizing agent, those type of break repair by homologous or non-homologous end joining when there is not any kind of temple present to fil the gap. Ifthis gap did not get fll cause fragmentation of chromosome. Non-Homologous End Joining: Broken end recognizes by Ku70/80 heterodimer because this dimer act as a sensor and Ku70/80 heterodimer binds to ‘those ends, then the bridge between those end form by MRN complex (in yeast MRX) contain Rad 50, Mre lland Nbsi (inyeast called Xrs2), The broken end also comes together because individual Ku protein has the affinity towards each other. Then Artemis protein gets activated by phosphorylation through DNA dependent protein kinase (DNA-PKcs), and now inactivated form act as endonuclease and exonuclease and responsible for trimming the overhangs. Both DNA-PKcs and Artemis start to act by the permission of Ku protein. After that, the gaps get filled by DNA polymerase and the end join finally by the DNA ligase IV in the alliance of XRCC4. Non-homologous end joining takes place throughout the cell cycle but specifically occur in G, and G,. For example, this mechanism visualizes in the case of recombination ofimmunoglobin gene. Due to this pathway change occurin DNA strands. Homologous end joining: Itis carried out by general homologous recombination mechanism, this mechanism takes place throughout the cell cycle but specifically occur in G, and S. Due to this pathway not any kind of change occurin DNA strands.Molecular Biology 298 Let's Talk Publication Procaryotic type Eukaryotic type Double treag break Double tread break sere ————— End binding. ° End binding. ° : ‘3 89 81s End processing End process Mx complete te ma = Nuclease domain complet DUA Pk coma ‘temic 86 r Gap filing Gap, ra Polymerase S eg 96GB Ligation "3 z rm EZ (Saccharomyces cerevasse) — guesanacaten (lemmas) Fig. : Non-Homologous End-Joining Recombination Recombination is defined as the process that involves rearrangement of genetic material which takes place by the ‘means of breaking of genetic material and then rejoining ofthat genetic material. In eukaryotes, crossing over which is known as a physical exchange of genetic material and occur in prophase | of meiosis, also govern by mechanics of recombination. In eukaryotes recombination also takes place during repair. n prokaryotes, recombination occurs in Conjugation, transformation, and transduction during the integration of transferred genetic material into the bacterial chromosome, Here we mention three classes of genetic recombination, first - homologous recombination, second -site-specifirecombination and third—ONA transposition. 2.8. Homologous recombination or General recombination Exchange of segments of genetic material between the homologous gene sequences (minimum size length of 9 homologous sequence is up to100 base pair or a >75 base pair which is necessary for recombination process) or homologous chromosome. During meiosis crossing over (occur in pachytene of prophase |) and accurate chromosomal segregation govern by homologous recombination, thus responsible genetic diversity which occurs between resulting gametes. In prokaryotes, homologous recombination occurs during conjugation, transformation, and transduction andin those processes genetic material get exchange reciprocally ‘There are certain models which explain the mechanism of homologous recombination: 1. Holidaymodel: Itisgiven by Robin Holiday in 1964, This model is also known as heteroduplex model because this model also explains the recombination between short homologous sequences between two heteroduplexes.Let's Talk Publication 299 Molecular Biology Firstly two homologous DNA molecule get to align with each other after that nickis introduce in both dsDNA at same Position, in turn strand next to nick get unpaired to its complementary strand and free to invade the near align dsDNA. at the same location where nick form and after invasion base pair with its homologous region within that dsDNA, as a result, this invasion create holiday unction (across) which isthe key recombination intermediate, this move is known as strand invasion. After this step movement of a holiday, junction takes place along the DNA whichis called as branch migration, during, the migration of holiday junction base pair within parental DNA strand get broken at the same time as identical base pair produces within the recombination intermediate. After this step holiday junction get cleave, which occur by cleaving the DNA strand within the holiday junction, this event is known as resolution of holiday junction and it takes place by two cleaving pattern occurs in different orientation, those pattern get visualize by study the chi form of Holiday structure or three dimensional structure of Holiday structure. aligned homologous x 8 c duplexes 2 » « ee! oS op cuplex strand invasion 2nd strand aststrand ot heteroduplex branch migration bybrie heteroduplex ee Fig. : Formation of heteroduplexMolecular Biology 300 Let's Talk Publication In one pattern cut takes place in those strands which do not get nicked during initiation of recombination process or cut takes place up-down and then joined by DNA ligase, in turn, resulted in products called splice recombination product or crossover product, here reassortment of genes that next to the recombination site also takes place. In second pattern cut takes place in that strand which gets nicked during the intial reaction or cut takes place left-right ‘and then joined by DNA ligase, in turn, resulted from products called patch or non-cross over a product A 8 c a > € Rotation of one molecule Planar molecule Two stands are nicked NA cleavage "splice" or rassover products "patch" or noncrossover products reassortment of flanking genes no reassortment a 8 c A 5 c Oe —_t 8 fo 8 c ee Fig. : Holiday modelLet's Talk Publication 301 Molecular Biology (One modification which is called Meselson ~ Radding modification which explain that the nick produce in one of two dsDNA (heteroduplex DNA) and the nicked strand had free end which invade the dsDNA at the homologous position and complementary base pair with its homologous region as a result form D loop by displacing one strand of the nation nicked dsDNA and displace strand get cleaved at the junction, between base-paired regions and its single- stranded region which result in the production of the heteroduplex. This modification explains the formation of two nicks in two dsDNA at the same position and the formation of heteroduplex by two dsDNA molecule which interact at the beginning of the recombination process. ase sansa 5 eS ————<-, —— ————_—_ : |=, | stand imasion , ee ¢ [7 See ———————————— branch mation F Snitemedite wih A {wo oar cons 5 a * —— XL —_ ——— =... Fig. : Meselson-Radding inodification Double-strand break model: Firstly double-stranded break is created in one of the two dsDNA called recipient by the endonuclease, in meiosis Spo11 protein act as an endonuclease. Spot (DNA topoisomerase like an enzyme) binds to S' end of the break. DBSs inmitoticcell get generated by DNA damage, mating switch in yeast and V(D)} recombination. In both cases 5’ attack by an exonuclease and in case of meiotic cell Spot get dissociate by the action of exonuclease, in turn, create free 3° overhangiin the assistance of helicase and this process is known as 5'-end resection. 3’ end invades the nation nickedMolecular Biology 302 Let's Talk Publication 29, dsDNA called donor at the homologous region, in turn, form complementary base pairing, asa result, form D loop by displacing the non-complementary strand and form heteroduplex, the free 3’ end act as a primer for the DNA synthesis and cause the extension of Dloop. ‘The point is known as recombinant point or branch point where the individual DNA strand of one dsDNA crosses the ‘other dsDNA. After this process branch migration takes place by the movement of recombinant joint or Holiday junction or branch point in either direction and its dependence upon the direction of strand displacement. Capture of second double strand break done when another side of the break get a pair by D loop due to the extension of D loop. Thus here also a holiday junction get form after the DNA synthesis along with break fling and gap sealing by DNA ligase, as a result two holiday junction get form, which resolves by cutting within the holiday junction, If both crosses over resolvation takes place in opposite way, result in the formation of cross over product and If both crosses over resolvation takes place in same way, resultin the formation of non cross over product. Homologous recombination in bacteria Inbacteria, homologous recombination requires a free 3" end, whichis provided by various mean but forhomologous recombination, this end processing is done by three recombination system name RecBCD nuclease/helicase, Rect, Recf system, from all these RecBCD nuclease/helicases also called Exonuclease V having highest importance. As the name shows RecBCD has three subunits along with both nuclease and helicase activity. RecBCD is bipolar helicase because RecBis3'toS’ helicase and RecD is to 3’ helicase. RecB also has nuclease activity. RecCrecognizes Chisite. Firstly binding of RecBCD takes place at nicked or broken (free) end after that unwinding of dsDNA occur by helicase action in ATP dependent manner through the movement of RecB, a slow helicase in 3'to5’ direction and RecD in 5! to 3! faster than RecB in presence of SSB protein, as a result, in front of RecB accumulation of ssDNA occur. Distortion within the activity of enzyme takes place when it gets to interact with crossover Hotspot Instigator (Chi) sequence (Chi 4,009 in no within E.coli chromosome), which posses consensus sequence 5’GCTGGTGG3' and takes place one time teach 5-10 kbp and get recognise by RecC and get bind on RecC, after interaction and recognition with Chi, RecC signal RecD for stopping of unwinding and simultaneously RecD signal RecB to cut DNA. RecB cut the strand with Chi, in turn release the 3’ end having strand, after cutting strand continuous unwinding caused by the enayme, asa resulta free 3’ and with terminal chi sequence get generated, which act as a site to which ReaA (homologous to meiotic Dme1 protein and RadS1 of eukaryotes) loaded by RecBCD enzyme (act as RecA loader), RecA assemble on that strand in cooperative manner as six RecA monomer per turn of the strand by the disassembly of SSB protein, this RecA protein-coated strand capable to cause invasion within the intact dsDNA, in turn form D loop which leads to the formation of Holiday junction by successive cleavage ofthe strand which get displaced. E.coli ruv genes product identify the holiday junction, Ruva firstly recognizes the holiday junction and bind at there with all four strands of DNA. RuvA sandwich the DNA through binding, in a tetrameric form on both side of DNA. A hexameric helicase RuvB bind to both DNA strand upstream to crossover and supply motor for branch migration, in turn specifically catalyze branch migration, both RuvA/B complex mediate branch migration with the rate of 10to 20, bbp/sec by the displacement of RecA, RecG also a helicase function in association with A/8. After branch migration get completed two RuvC protein replace the RuvA/B and resolve the holiday junction by its endonucleolytic activity. ATTG is an asymmetric tetranucleotide act as a hotspot for RuvC for resolvation of holiday junction and responsible to mediate the resolution by consideration of nick production takes place in which pair of strands, as a result, a formation of cross over or non-crossover product formation. RecBCD enters at 058 Recd (fast) Rect DNA unwinding & loop formation | Rec® (slow)Let's Talk Publication 303, Molecular Biology DNA unwinding & lec (fas | loop formation RecQ (ast) loop growth 3’ and cleaved by helicase activity loop maximum ena 5 Encountering the Chi site causes pause of complex ——— nits 31-5’ nuclease activity no longer, now opposite end cleave fast loop shortening 3 . +4 slow (under load) Fig. : Homologous recombination in bacteriaMolecular Biology 304 Let's Talk Publication 2.10. Site-specificrecombination: Recombination can also take place when there isa short region of homology is presently known asa spectficregion of homology responsible for recombination, this type of recombination called site-specific recombination and visualize during the A bacteriophage integration within E.coli genome. This recombination occurs due to the presence of att {attachment site) site on both A bacteriophage called attP contain 250 bp along with POP and in E.coli called att® to contain 23 bp along with BOB’. Both att site has 15 base pair central homologous sequence called ‘O'on to which recombination occurs in case of integration but in case of excision those att site known as attL and llR, Firstly integration is carried out by lambda integrase which is related to IB topoisomerase family and possess a conserved tyrosine residue thus also called tyrosine recombinase. Integrase require the assistance of IHF (integration host factor), which is a heterodimer of 20 kDa and responsible to cause bend in DNA by binding the ~20 bp sequence of attP site,in turn integrase binding site come close, which present on the DNA arm, as a result integration of the lambda genome into E.coli genome takes place. Through integration the attachment (att) site generate called attL or BOP’ and attR or POB’ and excision takes place due to reciprocal recombination takes place between this two att site by the function of integrase, IHF, and Xis which act as excisionase. Xis inhibit Integration but promote excision, Site-specific recombination done by recombinase enzyme family and this event requires for the integration of phage genome sequence into E.coli genome and during the excision of phage genome sequence from the E.coli genome. For integration integrase enayme is required which is known as Int in phage, Cre in phage P1 and FLP in yeast (cause inversion of a chromosome). Here we explain Site-specific recombination between lambda phage and E.coli. lambda phage possess two type of lfe cycle mode, first, lytic in which it resides within E.coli as independent chromosomal ‘molecule and second, lysogenic, in which lambda phage DNA integrates in E.coli chromosome called integration, as well as release from the E.coli chromosome called excision and thus site-specific recombination, require to carry out both event. al c. tt? cc Qa» 8 ey atte att Fig. : Site-specific recognition There are two families of conservative site-specific recombinases: The serine recombinases and the tyrosine recombinases. Fundamental to the mechanism used by both families is that when they cleave the DNA, a covalent protein-DNA intermediate is generated. For the serine recombinases, the side chain of a serine residue within the protein’s active site attacks a specific phosphodiester bond in the recombination site, This reaction introduces aLet's Talk Publication 305 Molecular Biology single-strand breakin the DNA and simultaneously generates a covalent inkage between the serine and phosphate at this DNA cleavage site. Likewise, for the tyrosine recombinases, itis the side chain of the active site tyrosine that attacks and then becomes joined to the DNA. crossover region recombinase recognition sites 5 5 secon S| ceavoge ofl our stands of vA (two from each duplex) 3-H 5 ED 3 = ons" 3.40 es Ey SS oH" swap DNA partners 210° Q— (is | ee’ 3 ——__ pons a 3-H a 3 ——— oH3" rejoining recombinase (R1 with R3 & R2 with RA)Molecular Biology 306 Let's Talk Publication Cleavage of top stand by recombinase subunits R1 &RB right top DNA stands from both top & bottom stands swap partner Holiday junction —__' ‘ol J cleavage of "bottom" strands bby R2 & RA subunits & switching of partner bottom strand exchange to finish recombination d SS eS seg EE SS eee Fig. : Conservative Site Specific recombinasesLet's Talk Publication 307 ‘Molecular Biology 3, W (i) (iy 0 (a) (0) i) (iii) ‘Transcription is the process of formation of a complementary RNA copy of DNA sequence, which acts as a template. RNA polymerase isa catalytic enzyme in transcription. RNA polymerase reads DNA segment and synthesizes RNA in S' t03'inonedirection. In RNAs, only m-RNA act as coding and other are non-coding RNA (e.g., RNA, tRNA, siRNA, snRNA, snoRNA, PiwiRNA, Cajal RNA, Xist RNA, Aist RNA, siRNA, tmRNA). Thus only mRNA proceeds for translation. So it shows, transcription is the first step of gene expression, DNA sequent which gets transcribe known as a transcription unit, which encoded various kind of RNA, including the component ofthe protein assembly process and Ribozymes, ‘Transcription Unit DNA segments that transcribed into RNA and other DNA sequences which are necessary for transcription. Intranscription unit3 componentarenecessary. Promoter RNA Coding region Terminator sequence. Coding and terminator region get transcribe by RNA-polymerase so both area trans element. pone omen PWDDWIVIVIRNIWIITTIIN os : ; 3 a eck tnnr Tee Fig. : Transcription of coding and terminator region Promoter: Itisa binding site for RNA-polymerase, so transcription can begin. Promoter determines the Direction of transcription From two strand of DNA which strand read asa template by RNA pol. ‘The transcription startsite, also define the addition of the first nucleotide to RNA. DNA ()strand 5! 3 (+) strand 3 3 wa SIA 8 And is transcribed rom the (.) strand, Fig. : Transcription of different genes from different strands Coding region: Sequence positioned between promoter and terminator site which is copied intoa RNA. Coding region starts froma start site and finishes at the termination sequence. Thus the first nucleotide that is transcribed into RNA present in start site. Sequence present prior to starting a site called upstream (itis written as negative numbers like “1, -2,-3...). And sequence Present after start site called as downstream (itis writing positive numbers like +1, +2, Bu) ‘Terminator sequence: Itis a part of a coding region When RNA pol reaches terminator sequence the transcription is stopped,Molecular Biology 308 Let's Talk Publication 3.2. 0 i) (ii) w 0 (i) iy w) Only one strand of DNA is transcribed into RNA and this strand known as template strand or non-coding strand or antisense strand, And the second strandis called as a non-template or sense strand or coding strand. In double strand DNA which stand is transcribed into RNAis dependent on the position of a promoter, Prokaryotictranscription Ultrastructure of a prokaryotic promoter The promoter of bacteria consists3—4 consensus sequence. Pribnow box (-10 sequence) Recognition box (-35 sequence) uPelement. “10 extended box Promoter Nontemplate oat strand — Sa “eral Ts 7 emplate Consensus Consensus Transeription | sand sequence sequence startsite Fig. : Ultra Structure of Prokaryotic Promoter Pribnow box: Itpresentin all prokaryotic promoter. Itis a6 bp region A:T rich DNA and located at upstream of the start point. The hexameric sequence is TATAAT, The center Tof this sequence present at ~ 10 positions from start site thus name is~ 10 sequence. In TATAAT sequence, of first Tat first position is 80%, the chance of A at the second position is. 95%, a chance of Tat third position is 45%. So this sequence can be summarized a T80 AS T45 AGO ASOT96. Initial TA ishighly conserved and final Tis completely conserved. in promoter, Pribnow boxis present in Z-DNA form because of diameter of Z-DNAis less than B-DNA, Recognition box : (-35 sequence): It is located at -35 positions in upstream of the start point. Itis a 6bp region. This, box also called a GC box. The consensus is TTGACA (T82T84G78 AGS C54 ASA), Generally, the spacer present between Pribnow box and recognition sequence is 16 ~18 bp but in some cases as small as 15 bp or as large as 20 bp observed with an exception. The length of spacer significant for correct placing of RNA polymerase due to their geometry. UP elements: (between — 40 and —60) Its present at several bp farther in upstream of - 35 box. tis nat present in all promoter (some strong promoter contain up elements). Subunit of RNA polymerase bind which it. e.g., ‘NA promoter contains up elements. -10 extended box: upstream to -10 box. If -35 box absent, then responsible for compensation of -35 box. e.g., Gal gene of Ecol. Type of promoteron the bases of workefficiency Strong Promoter: Strong promoter has a high affinity for RNA polymerase. The transcription rate of the gene associated with the strong promoter is very high. A promoter having more number of a consensus sequence or having sequence which show close homology to the consensus sequence. Potential of strong promoter depend upon some characteristic like how eagerly promoter allows RNA polymerase to escape it, a number of transcription initiate at a time and how capableit initiate isomerization (change in the structure of RNA pol) Weak Promoter: Weak promoter has less affinity RNA polymerase. The transcription rate of the gene associated with the weak promoter is very log. Walk having less number of a consensus sequence or having sequence show less homology tothe consensus sequence.