FACULTY OF MEDICAL SCIENCES

Prepared by: Priyancka Arora, Assistant Professor, Department of Biotechnology, Rama

Univeristy

DNA Repair
It refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule.

Importance of repair system:

DNA is molecule that plays an active and critical role in cell division and is transmitted through
generations, so this should not be mutated, as if mutated, will lead to mutations and genetic diseases.

The mechanisms used by cells to do so includes:

1. Proofreading
2. Direct repair
3. Excision repair
4. Mismatch repair
5. Recombinational repair
6. Repair of double strand DNA break
7. SOS/ Transleison repair

I. Proofreading

During DNA replication, different types of polymerase enzymes are involved, which other than
polymerase activity, also perform exonuclease activity and they themselves check their work, which
each base that they add. This process is called proofreading.

II. Direct Repair

Act directly on damaged nucleotides and reverses it to the normal form (Direct reversal).
Simplest repair mechanism- Binds to the damage and restores the DNA genome to its normal
state in single reaction step.

Example:
a) Pyrimidine Dimer Repair:
 Pyrimidine dimers are the molecular lesions formed from thymine or cytosine
bases in the DNA via photochemical reactions.
 Pyrimidine dimers are formed because of UV rays.
 Ultraviolet light induces the formation of covalent linkages by the reactions
localized on C=C double bonds.
 These lesions alter the structure of DNA and consequently inhibit polymerases
and arrest replication.
 It is repaired by light- dependent photoreactivation.
 DNA photolyase are the enzymes responsible for repairing pyrimidine dimers.
 Light of wavelength 300
300-500
500 nm is used to stimulate enzymes, which then binds
to the pyrimidine dimer and convert tthem
hem back to the original form.
b) O6- methylguanine repair-
 Guanine can undergo a reaction that attaches a methyl (-CH3) group to an
oxygen atom, in the presence of alkylating agents, in the base. This is called O 6-
methylguanine.
 This methyl-bearing guanine formed has tendency to pair with Thymine (T)
instead of cytosine ©, during chain synthesis.
 O6- methylguanine DNA methyl transferases ia an enzyme that can remove the
methyl group and reverses the base to normal form.
 Now again Guanine will tend to bind/ pair with cystine.
III. Excision Repair:
 Corrects Damage to one or a few bases of DNA.
 Repaired by excision/ removal of damaged region.
 Then re-synthesis of correct sequences by DNA polymerase at the damaged site, which
is now removed.
 There are two types of Excision repair: Base excision repair and Nucleotide Excision
repair
a. Base- Excision Repair
 It involves removal of damaged base.
 DNA Glycosylase enzymes initiates this process of repair
 8 genes encoding different DNA glycosylase are present, where each one Is responsible
for identifying and repairing special kind of base damage.
 DNA glycosylase cleaves N-glycosidic bonds and release the damaged base, thus
generating an apurinic or apyrimidic site (AP Site)

Steps:
  Eg of Mutation - Spontaneous Deamination i.e. conversion of C to U or Oxidation of
guanine i.e. conversion of Guanine to oxo-guanine.
 Firstly, damage is recognized and Uracil DNA glycosylase gets activated and removal
of the mutated base from the DNA helix. DNA glycosylase breaks the glycosidic bond
between sugar and the faulty base.
 Next, an enzyme called AP (apurinic/apyrimidinic) endonuclease makes an incision
at the AP site, creating a break, or nick, at 5’ end of the AP site, at the strand of
DNA.
 Enzyme Lyase/AP exonuclease, cleaves the sugar-phosphate backbone containing
the AP site.
 Correct base pairs are incorporated by DNA polymerase I(Prokaryotes) or DNA
polymerase β (Eukaryotes).
 The phosphodiester bond is sealed by DNA ligase.

b. Nucleotide- Excision Repair

 In this type of excision repair, it removes DNA upto 30 bases in length.
 Even though there may be only a single incorrect base to be corrected, but it act upon
whole damaged area of DNA.
 A patch/Stretch of DNA is removed.
 It breaks phopsphodiester bond on either side of the lesion, thus excision of an
oligonucleotide.
 This is more complex than base excision repair, especially in Eukaryotes.
 The damage is recognized by one or more proteins that assemble at the location.

Steps:

1. Recognition of damaged site.

2. Unwinding (Helicase) to form a bubble.
3. Oligonucleotide excision.
4. DNA polymerase replaces the missing stretch of DNA and DNA ligase will seal the gap.

Prokaryotes:

The Uvr system of NER in E. Coli:

1. There are 3 proteins called as Uvr A, Uvr B and Uvr C, which is involved in damage
recognition and endonuclease excision.
2. Uvr A+ Uvr B binds at damage site. Uvr A recognizes damage.
3. After recognistion, Uvr A is removed and allows Uvr C to bind.
4. Uvr BC- cleaves at 8Th phosphodiester bond on 5’ end of the lesion and at 4th/5th on 3’
end.
5. This fragment is released by Uvr D (Helicase), as it helps in unwinding the DNA to allow
release of single strand between the two cuts created.
 6. There is a removal of usually 12-13 nucleotide in length DNA.
7. The gap which is generated is finally submitted for repair synthesis, with the use of DNA
polymerase I and ligase.

Eukaryotes:

Protein Function Eukaryotic Enzymes Prokaryotic enzymes

Detection of damage XPC UvrA
Helicase XPA and XPD Uvr D
3’ end endonuclease XPA Uvr BC
5’ end Endonuclease ERCC1-XPF Uvr BC

Problems due to defective NER system:

1. Xeroderma Pigmentosa (XP)- In humans

 XP is rare inherited disese.
 Pigmented lesions on areas of skin exposed to sun.
 Elevated level of skin cancer
  Inability to repair DNA-lesions induced by UV rays.
2. Cockayne syndrome- caused by genetically defective NER system.
3. Trichothiodystrophy

IV. Mismatch Repair

 Plays essential role in the maintenance of genetic information.
 It enhances genomic stability by correcting errors that have escaped proofreading.
 It can discriminate between new strand from parental strand, as only new strand needs
repair.
 When two bases are mispaired, how the proteins involved in mismatch repair
distinguish between right and wrong base?
In bacteria, the parental strand (old) and daughter (new) strand are distinguish by
methylation state. An old strand will have methyl group attached to some of its bases,
but since new strand is just synthesized, so methylation is not yet started.

Methylation in E.Coli

 In E.coli, the methylation of A in GATC motif is done, which provide a marker for a repair
system to differentiate between parent and daughter strand.
 Dam methylase (DNA Adenine methylase), catalyses the transfer of a methyl group (-
CH3) to A of the 5’ GATC 3’ sequence in duplex DNA.
 This step is delayed for several minutes after replication, which allows the mismatch
repair system to do its work.
 Dam converts Adenine to 6-methyladenines.

Enzyme system involved in mismatch repair:

1. Recognizes mismatch pairs

2. Determines which base to remove
3. Excise incorrect base
4. Repair synthesis
5. Sealing the gap

Mismatch repair in E.Coli (Proteins involved):

1. MutS is responsible for initiation of mismatch repair. MutS is a 95 KDa polypeptide,

exists as dimmers and tetramers. It recognizes mismatched base pairs. It bind to the
DNA mismatch as a homodimer complex and recruits MutL.
2. MutL and MutS heteroduplex is believe to be a key intermediate in the initiation of
repair system.
3. MutH proteins cleaves the unmethylated strand on the 5’ side of the G in GATC
sequences.

Steps of Mismatch Repair in E.Coli:

1. MutS will recognize mismatch pair of base and bind at that site in DNA>
2. MutL is recruited to the site.
3. MutH is present at nearby GATC sequence in its inactive form. When in contact with
MutL, it gets activated.
4. MutH then gets activated in the presence of ATP at a hemimethylated GATC. It then
cleaves the unmethylated DNA strand, leaving a nick at 5’ to G of GATC sequence.
5. MutU acts a helicase and unwind the strand.
6. The exonuclease then translocates and digests the nucleotide sequences till it reaches
the other side of the mismatch (3’).
Exonuclease that acts in 5’ to 3’ direction is called Rec J
Exonuclease that acts in 3’to 5’ direction is called exonuclease I
7. This forms a gap, which is filled by the action of DNA polymerase I and DNA ligase then
help to seal the gap.
V. Recombinational repair ( Retrieval system)
 When excision repair mechanism fails, recombinational repair operation starts to repair
errors.
 The DNA synthesis immediately after UV irradiation in excision repair deficient cells has
discontinuities in strand synthesis. (also in normal cells)
 When DNA synthesis proceeds along a damaged template, synthesis halts at the site of a
non-coding lesion, and then resumes downstream from the lesion, thus leaving a gap in
the newly synthesized daughter strand opposite to lesion in the parental strand.
 These gaps are no longer subjected to excision, since this process requires an intact
complementary strand.
 So, it is required to fill the gap first.
 These gaps in the daughter strand are subjected to repair by transferring the
appropriate sections of DNA from the lagging DNA parental strand.
 That is why name retrieval system was given, as gap is filled in one strand of dsDNA by
retrieving a homologous single strand from another dsDNA.
 It is completely dependent on Rec A protein.

Steps in Recombinational repair:

1. Replication is halted at the mutation site, leaves a gap.

2. Rec BCD (Nuclease and helicase) binds the site of gap. Nuclease helps in cutting the
complementary strand from sister duplex, which will help in filling the gap formed opposite to
the lesion.
3. Rec A along with Rec FOR binds and helps in invasion of strand to its sister duplex. Rec A
mediates the process of invasion. Rec FOR promotes Rec A single strand DNA filament formation
& avoid Rec A loading defects.
4. Ruv ABC- Ruv A&B catalyses branch migration.
Ruv C binds specifically to the holliday junction as a dimer and uphold or resolve the junction
into open- planar configuration.
5. Now, the parental damaged strand faces the normal strand.
6. Since, the damage now lies opposite to a normal strand and therefore, can be dealt with
excision repair system. As now, the gap is filled and excision repair system can function.
VI. Double strand DNA break repair (DSB repair)
 Double stranded brea breakk in DNA can result in loss and rearrangement of genomic
sequences that is why it is needed to be repaired.
  It is not formed as a consequence of the UV-radiation absorption, rather, they are
formed as the consequences of the attempted repair of UV-induced base damage in
DNA.
 There are two pathways in the repair of double-stranded DNA break:
Non-homologous end joining
Homologous end joining

Non-Homologous end joining (NHEJ)

 Major DNA DSB repair pathway in human cells due to ionizing radiation.
 Doesn’t require DNA template.
 Active throughout the cell cycle.

Proteins involved in NHEJ:

1. Ku heterodimer:
 In prokaryotes: Ku 30-40 KDa
In Eukaryotes: Ku 70-80 KDa
 DNA binding protein
 ATPase activity
 Helicase activity
 Helps in recruiting other proteins to damaged site.

2. DNA-PK
 It phosphorylates serine/ threonine preceded by glutamine at damaged site.

3. Artemis
 Substrate for DNA-PK
 Binds to DNA-PK and gets phosphorylated by it.
 It also auto-phosphorylates.
 Nuclease (cleaves the end of damaged DNA to prepare it for ligation)

4. DNA Ligase IV
 End processing enzyme that assist in DNA end ligation.

5. XRCC4 (X-ray repair cross complementing protein 4)

 It works in association with DNA ligase IV in the ligation of ds DNA molecule.
 It enables the interaction of DNA ligase IV to damaged DNA.

6. DNA polymerase β
 Repair synthesis by proliferating the nucleotide sequences.
 7. Polynucleotide kinase
 Transfer phosphate group from ATP to 5’-OH (kinase)
 Remove phosphate from 3’ phosphate group (phosphatase).
 Thus converts non-ligatable nick to ligatable (5’ phosphate – 3’OH) nick.

Steps of NHEJ :

1. Recognition of damaged ends by heterodimer Ku 70/80.

2. Ku protein recruits DNA-PKs. DNA-PK can bind to damaged end even in the absence of Ku, but
Ku increases the affinity by 100 folds.
3. Artemis remains bound to DNA-PK even in the absence of DNA-damaged ends. So along with
DNA-PKs, artemis also gets recruited to damaged site.
4. DNA-PK after binding to the damage end gets activated and phosphorylates artemis and itself.
5. Artemis starts endonuclease activity and cut away ssDNA overhangs at ends.
6. XRCC4 and DNA ligase IV gets recruited by Ku and polynucleotide kinase and polymerase gets
recruited by XRCC4.
7. Ligation of 2 strands.

Homologous End joining:

 Activated when the cells is in late S/G2 phase of the cell cycle and the template has recently
been duplicated (sister chromatid).
 Requires the presence of an identical or nearly identical sequence to be used as template.
 Homologous recombination repair is a DNA repair process that includes the invasion of an
undamaged DNA molecule by a damaged molecule of identical or very similar sequence.
Resynthesis of the damaged region is accomplished using the undamaged molecule as a
template.
 Sister chromatids (duplicated chromosomes following DNA replication)
or the paternal and maternal copies of chromosomes provide the required
homology (sequence identity or near-identity over a few hundred DNA
base pairs).
 

Steps:

 Double stranded damage.

 5’ to 3’ resection (nuclease & helicase enzymes)
 Strand invasion
 New strand synthesis.
 Formation of holliday junction
 Resolution of junction
 Healing of DSB in DNA

Proteins required for homologous end joining DSB DNA repair

E.coli Eukaryotes Function
Rec J, BCD MRN complex Nuclease & helicase
Rec A Rad 51 Strand invasion
Ruv A - Complex with holliday junction
Ruv B - ATPase, branch Migration
Ruv C XRCC3 complex Endonuclease, resolution of
holliday junction
VII. SOS response/ Translesion repair
 The SOS response is the term used to describe changes in gene expression in E. coli in
other bacteria in response to extensive DNA damage. The prokaryotic SOS system is
regulated by two main protien i.e. Lex A and Rec A.
  •Despite having multiple repair system, sometimes the damage to an organism’s DNA is
so great that the normal repair mechanisms just described cannot repair all the damage.
As a result, DNA synthesis stops completely. In such situations, a global control network
called the SOS response is activated.
 RecA–ssDNA filaments activate LexA auto protease activity, which ultimately leads to
cleavage of LexA dimer and subsequent LexA degradation.
 The loss of LexA repressor induces transcription of the SOS genes and allows for further
signal induction, inhibition of cell division and an increase in levels of proteins
responsible for damage processing.
 The LexA homodimer is a transcriptional repressor that binds to operator sequences
commonly refers to as SOS boxes. In E. Coli it is known that Lex A regulates transcription
of approximately 48 genes including LexA and RecA genes
 lexA negatively regulates the function of many genes involved in DNA repair and
synthesis. Destruction of LexA increases transcription of genes for excision repair.
 LexA is also involved in formation of UvrB protein, required in nucleotide excision repair.

The SOS regulatory system

 LexA repressor, which inhibits expression of the SOS genes during normal cell growth
 RecA protein, which is activated by treatments that turn on the SOS response
 The lesion repair genes are induced at the beginning of SOS response. The error-prone
translesion polymerases, for example, UmuCD’2 (also called DNA polymerase V), are
induced later on as a last resort. Once the DNA damage is repaired or bypassed using
polymerases or through recombination, the amount of single-stranded DNA in cells is
decreased, lowering the amounts of RecA filaments decreases cleavage activity of LexA
homodimer, which then binds to the SOS boxes near promoters and restores normal
gene expression.

SOS Mechanism:
 During normal growth, the SOS genes are negatively regulated by LexA repressor
protein dimmers. Under normal conditions, LexA bind to a 20-bp consensus sequence
(the SOS box) in the operator region for those genes.
 Some of these genes are expressed at certain levels in the repressed state, according to
the affinity of LexA for their SOS box.
 Activation of SOS genes occurs after DNA damage by the accumulation of single-
stranded (ssDNA) regions generated at replication forks, where DNA polymerase is
blocked.
 RecA forms a filament around these ssDNA in an ATP-dependent fashion, and become
activated.
 The activated form of RecA interacts with the lexA repressor to facilitate the lexA
repressor’s self cleavage from the operator.
 Once the pool of lexA decreases, repression of the SOS genes goes down according to
the level of LexA affinity for the SOS boxes.
 Operators that bind weakly LexA are the first to be fully expressed.
 In this way LexA can sequentially activate different mechanism of repair.
 Genes having a weak SOS box( such as lexA, recA, UvrA, UvrB and UvrD) are fully
induced in response to even weak SOS-inducing treatments.
 Thus the first SOS repair mechanism to be induced is nucleotide excision repair(NER),
whose aim is to fix DNA damage without commitment to a full-fledged SOS response.
 If, however, NER doesn’t suffice to fix the damage, the LexA concentration is further
reduced, so the expression of genes with stronger LexA Boxes (such as UmuD, UmuC) is
induced.
 Upon cleavage by RecA, UmuD form UmuD’. Two UmuD’ along with One UmuC
combines to form DNA polymerase V, known as translesion DNA polymerase. It doesn’t
contain proofreading activity, thus the polymerization activity is error-prone.
 SOS repair system operates only under potentially lethal conditions caused by extensive
UV irradiation. Therefore, SOS repair system works on the principle that survival with
mutation is better than no survival at all. This repair is also called Error prone repair or
translation replication.