Mechanism of replication Semiconservative as studied in prokaryotes Replication involves separation of the two original strands and synthesis of two

wo new daughter strands using the original strands as templates Semiconservative replication: each daughter strand contains one template strand and one newly synthesized strand Direction of replication DNA double helix unwinds at a specific point called an origin of replication Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is bidirectional in most organisms At each origin of replication, there are two replication forks, points at which new polynucleotide chains are formed There is one origin of replication and two replication forks in the circular DNA of prokaryotes In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin DNA synthesis based on two template strands: leading strand and lagging strand templates; mechanism in prokaryotes is presented DNA is synthesized from its 5 -> 3 end (from the 3 -> 5 direction of the template) the leading strand is synthesized continuously in the 5 -> 3 direction toward the replication fork the lagging strand is synthesized semidiscontinuously (Okazaki fragments) also in the 5 -> 3 direction, but away from the replication fork lagging strand fragments are joined by the enzyme DNA ligase Enzymes and proteins in DNA replication

Other requirements: NTPs (Primer RNA) dNTPs and Mg+2

Timothy John S. Bautista 2A Pharmacy

University of Santo Tomas Biochemistry Lecture

Faculty of Pharmacy NA: Central Dogma

Replication fork

Timothy John S. Bautista 2A Pharmacy

University of Santo Tomas Biochemistry Lecture

Faculty of Pharmacy NA: Central Dogma

Exonuclease activity: Remove primers; remove mismatched bases

a) 3 to 5 exonuclease activity (DNA POL I and DNA POL III) b) 5 to 3 exonuclease activity (DNA POL I) Replication Unwinding DNA gyrase introduces a swivel point in advance of the replication fork a helicase binds at the replication fork and promotes unwinding single-stranded binding (SSB) protein protects exposed regions of single-stranded DNA; stabilizes the single strands Primase catalyzes the synthesis of RNA primer Synthesis catalyzed by Pol III primer removed by Pol I DNA ligase seals remaining nicks *DNA replication occurs during the S phase in eukaryotes *Telomerase enzyme that extends the 3OH of replicated DNA in eukaryotes as primer is removed from leading strand; Telomeres shorten during normal cell cycle Changes in DNA sequence (mutation) Point mutations: transition (purine to purine alteration; pyrimidine to pyrimidine , transversion pyrimidine to purine or vice versa; frameshift mutation alteration in the reading frame Silent mutation no change in the function of the protein products Spontaneous changes that occur during normal and metabolic function of the cell Induced environmental factors

Timothy John S. Bautista 2A Pharmacy

University of Santo Tomas Biochemistry Lecture

Faculty of Pharmacy NA: Central Dogma

Spontaneous mutation Replication errors Mismatching of base pairs 1) Base substitution- due to tautomerization of the bases; lead to non standard base pairing 2) Removal of purine by hydrolysis of the N-C glycosidic bond 3) Deamination of the base (cytosine to uracil) Insertion of one or more base pairs Deletion of one or more base pairs DNA Repair mechanisms 1) Most common mismatched repair mechanism Enzymes in repair mechanisms - hydrolysis of phosphodiester bond by endonuclease - exonuclease removes the mismatched nucleotide -DNA polymerase catalyzes the addition of the correct nucleotide - gap is closed by DNA ligase 2) DNA Glycosidases remove deaminated base; endonuclease removes the sugar; DNA Pol I puts in the correct nucleotide; gap closed by DNA ligase RNA synthesis (transcription) Transcription Template is DNA Major enzyme: DNA directed RNA polymerase No need for primers 5 to 3 direction Requires a promoter region in the template DNA to which the RNA polymerse will bind Promoter 40 base pairs upstream away from the start site (+1) (-10 bases Pribnow box (TATAA box and -35 with sequence TTGACA) Three stages: initiation, elongation, termination Termination may be rho factor dependent rho factor terminates synthesis or rho factor independent formation of a stable hairpin loop

Timothy John S. Bautista 2A Pharmacy

University of Santo Tomas Biochemistry Lecture

Faculty of Pharmacy NA: Central Dogma

Step1 -recognition of promoter by sigma factor; binding of polymerase holoenzyme to DNA; migration to promoter Step2 -formation of RNA polymerase; closed promoter complex Step3 -unwinding of DNA at promoter and formation of open promoter complex Step4 -RNA polymerase initiates mRNA synthesis; almost always with purines Step5 -RNA polymerase holoenzyme-catalyzed elongation of mRNA by about 4 or more nucleotides Step6 -Relese of alpha-subunits as core-RNA polymerase; proceeds down the template, elongating RNA transcript 3 types of RNA in eukaryotic transcription RNA pol I transcribes large ribosomal RNA genes RNA pol II transcribes protein encoding gene RNA pol III transcribes small RNAs (including tRNA and 5SRNA) *Post transcriptional modification of eukaryotic t-RNA: trimming of the 5end; removal of a fragment at the 3OH; addition of ACC at the 3OH Post transcriptional modification of eukaryotic m-RNA Capping methyl guanosine attachment at the 5 end to protect the cleavage of the RNA by exonucleases as RNA moves out of the nucleus Addition of poly A at the 3 end (20-250 long) helps to stabilize the mRNA structure; increases resistance to cellular nucleases Splicing removal of non coding sequences (introns)

Timothy John S. Bautista 2A Pharmacy

University of Santo Tomas Biochemistry Lecture

Faculty of Pharmacy NA: Central Dogma