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The following points highlight the top six repair mechanisms of damaged DNA. The repair mechanisms are: 1. Photo Reactivation 2. Excision Repair 3. Re-Combinational Repair Mechanism 4. Repair of DNA by Homologous Recombination 5. SOS Repair of Damaged DNA 6. Mismatch Repair.
Repair Mechanism # 1. Photo Reactivation:
We know that exposure of UV-irradiated bacteria immediately afterwards to visible light restores to a considerable degree the viability of the UV-inactivated bacteria. This phenomenon mown as photo reactivation, is based on enzymatic cleavage of the thymine dimers.
The enzyme, photolyase, binds to the thymine dimer and catalyses photochemical cleavage of the cyclobutane ring of the dimer to make the thymine’s free. The enzyme uses visible light for the reaction. Besides thymine-dimers, other pyrimidine-dimers—like cytosine-cytosine and cytosine-thymine dimer—are also attacked by the enzyme. The enzyme is devoid of any species-specificity. Photolyases have been detected in both prokaryotes and eukaryotes.
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The action of photolyase on UV-irradiated DNA containing thymine dimers is schematically represented in Fig. 9.70:
It has been observed that UV-irradiated DNA containing 5-bromouracil — which is an analogue of thymine and is incorporated into replicating DNA replacing thymine — is resistant to photo reactivation. Such DNA binds the photolyase enzyme, but the enzyme neither dissociates from the dimer nor can liberate the free thymine molecules.
Repair Mechanism # 2. Excision Repair:
Apart from photo reactivation, there are also other mechanisms for repair of damaged DNA. One of these is excision repair which occurs in absence of light i.e. exposure to visible light is not required. It is also known as dark-repair.
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The exicision repair essentially consists of removal of a segment of DNA containing the damaged portion of one strand of DNA and new synthesis of the removed segment of the DNA strand using the undamaged strand as template. The first step, known as incision step, involves recognition of the damaged segment by an endonuclease.
The enzyme cleaves the phosphodiester bond of the sugar- phosphate backbone at the 5′-end about eight nucleotides ahead of the damaged site producing a 3′-OH group free. The next step, known as excision step, involves a cut at a site 4 to 5 nucleotides downstream from the damaged site catalysed by a 5′-3′ exonuclease.
Thereby, a segment of DNA including the damaged site is removed. In the last step, known as the repair step, DNA polymerase I synthesizes a new stretch of DNA strand starting from the 3′-OH end using the intact complimentary strand as template. Finally, the newly synthesized strand is joined with the 5′-end by DNA ligase to complete the repair. The steps are diagrammatically shown in Fig. 9.71.
Another variation of excision repair is catalysed by the enzyme glycosylase which cleaves the N-glycosidic bond between a thymine of the thymine dimer to the sugar-phosphate backbone of the DNA strand. At the next step, the phosphodiester bond is cleared by the endonuclease activity of the same enzyme which recognizes a blank deoxyribose without a base attached to it.
In the following step, DNA polymerase I initiates DNA synthesis at the free 3′-OH end displacing the thymine dimer along with a few more adjacent nucleotides as shown in Fig. 9.72. This type of excision repair occurs also in E. coli and several other bacteria like Micrococcus luteus.
Excision repair is observed when UV-treated bacteria are stored in dark for a few hours in a medium which does not support growth before returning them to the normal growth supporting medium (liquid holding recovery). In excision repair mechanism DNA polymerase I seems to play an important role.
It has been shown that E. coli mutants which are deficient in this enzyme show extensive DNA damage following UV-irradiation, presumably because such mutants are unable to repair the damaged DNA by excision repair mechanism. It may be reminded that in normal DNA replication, polymerase III (pol III) catalyses DNA synthesis.
Repair Mechanism # 3. Re-Combinational Repair Mechanism:
This is another mode of repair of damaged DNA. It consists essentially of an exchange of a damaged segment of one DNA molecule by an undamaged segment of another one. As such exchange takes place only after replication of the damaged DNA has taken place it is also known as post- replication repair.
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When a damaged DNA molecule — e.g. DNA containing thymine-dimers induced by UV-irradiation—begins replication with the help of DNA polymerase III, the enzyme stops synthesis as it reaches a dimer, because of the distortion caused by the dimer in the regular double helix.
As a result, the progress of the replication fork halts temporarily as it reaches a dimer. DNA synthesis is then reinitiated at a new site, few nucleotides past the thymine-dimer site. Thus, a gap is created opposite the dimer site and a few adjoining nucleotides. The newly synthesized daughter strand is produced with several gaps i.e. in short pieces, if several dimers occur in the same template strand (Fig. 9.73).
The gaps in the daughter strand are then filled up by exchanging undamaged homologous segments from a sister DNA double helix. The gaps produced in the donor strand are then filled up by new DNA synthesis with the help of DNA polymerase I and sealed by the DNA ligase as shown in Fig. 9.74. Thus, in re-combinational repair system, parts of DNA strand missing in one strand are retrieved from another normal DNA strand of a sister double helix.
The damaged DNA strand will continue to have the damaged site and will replicate to have gaps in the daughter strand which will be repaired by re-combinational repair mechanism. Ultimately, the damaged strand will be outnumbered by normal DNA and will be insignificant.
In re-combinational repair mechanism, recA gene plays an important role. It has been observed that recA mutants are extremely sensitive to lethal effects of radiations and chemical mutagens. The recA gene is known to play a very important role in genetic recombination e.g. in conjugation where recA’ recipient fails to show genetic recombination. The gene functions also in recombinational repair, where exchange of DNA strands is involved.
Repair Mechanism # 4. Repair of DNA by Homologous Recombination:
This type of repair is called for when both strands of DNA molecules are damaged at sites opposite each other. In such a case, the missing segments cannot be replaced from sister strands after replication by the usual recombinational repair mechanism. The lost portions have to be retrieved from another homologous DNA molecule.
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In actively growing bacteria, each cell contains generally more than one copy of DNA. So, the lost portions of one damaged molecule can be repaired by crossing-over with a normal DNA molecule involving exchange of homologous segments. This type of DNA repair is known as homologous recombinational repair. Double-stranded breaks of DNA can be induced by exposure to X-rays. Rec A protein plays an important role also in this type of repair.
The repair process begins with the production of single-stranded segments at the 3′-OH end of each strand through the action of a nuclease and binding of the Rec A protein to the single strands. The binding of Rec A protein initiates strand exchange between homologous segments of the normal DNA duplex and the damaged one. The crossing-over results in the formation of two DNA molecules, each having an intact strand and a strand with gaps.
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The gaps are then filled up by new DNA synthesis catalysed by DNA polymerase and sealing by DNA ligase. The intact strand is used as template. Thus, crossing-over which is normally a mechanism for creating genetic diversity by mixing up genes located on homologous chromosomes, can also function as a means for repairing damaged DNA. Rec A protein plays vital roles in both the processes (Fig. 9.75).
Repair Mechanism # 5. SOS Repair of Damaged DNA:
The SOS repair mechanism functions in a more complicated way. Damages inflicted on DNA by mutagenic agents induce a complex series of changes which are collectively known as SOS response. The response leads to increased capacity to repair damaged DNA by excision repair and recombinational pathways.
The SOS response is set in action by the interaction of two proteins, — Rec A protein which is a product of the recA gene and Lex A protein, the product of lexA gene. The Rec A protein in addition to having a role in genetic recombination and recombinational repair also has a protease function. The Lex A protein acts as a repressor for a number of genes, known as SOS genes including the recA gene. Under normal conditions i.e. when the SOS response is not necessary, these genes remain repressed by the Lex A repressor.
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The initial event in the SOS response is the activation of RecA protease activity induced by DNA damage. The activation of Rec A protease activity occurs within a few minutes of DNA damage. The protease activity catalyses cleavage of the Lex A repressor making it inactive. As a result, the SOS genes can now be expressed to produce the enzymes required for DNA repair.
The events are shown in Fig. 9.76:
The SOS response, as the name suggests, is an emergency measure to repair mutational damage. It makes it possible for the cell to survive under conditions which would have been otherwise lethal. However, the possibility of generating new mutations increases in the repair of DNA molecules. This is because the SOS repair system allows DNA synthesis bypassing the damaged site.
When the DNA polymerase III reaches a damaged site to which Rec A binds, the protein (Rec A) interacts with the epsilon subunit of the DNA polymerase molecule. This subunit is responsible for insertion of the correct base into the growing DNA strand. As a result, chain elongation continues bypassing the damaged site, but the chance of incorporation of a wrong base increases. SOS repair, therefore, enhances the chance of mutation due to mis-pairing of bases. This is known as error-prone bypass repair.
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A more recent model based on SOS repair of UV-irrediated DNA in bacteriophages has been proposed. UV-irradiation is known to produce dimers of not only thymine, but also of thymine and cytosine and cytosine. During replication when 
are reached, the SOS repair system is halted temporarily and cystosine is deaminated to uracil.
Uracil pairs with adenine, bringing about a transition mutaition by changing C-G base pair to T-A. This is an error-free bypass repair thought it still causes a mutation. It is called error-free because the template DNA strand is faithfully copied in the newly synthesized strand. The change from C to U occurs in the template strand itself.
Repair Mechanism # 6. Mismatch Repair:
The rate of mutation varies usually in the range of 10-7 to 10-11 in bacteria. However, during normal DNA replication an error in inserting a correct base in the new strand occurs at a much higher rate, generally at a frequency the of 10-5. This large difference indicates that bacteria possess an in-built mechanism to rectify most of errors during replication.
Most bacterial DNA polymerases, in addition to having the polymerase activity, have also an exonuclease activity which works in the opposite direction i.e. while polymerization proceeds in the 5′ —> 3′ direction, the exonuclease works in the 3′ —> 5′ direction. Whenever a wrong base is inserted into the polynucleotide chain by mistake, the DNA polymerase stops and goes one step backward and the incorrect base is removed by the exonuclease activity. The polymerase then resumes its normal activity by inserting a correct base. This is known as the proof-reading function of the DNA polymerase. Mutants with an altered epsilon subunit of the DNA polymerase fails to perform the proof-reading function.
Although proof-reading by DNA-polymerase is an efficient way of removing many mismatched bases, a number of such errors may still persist after replication. Such mismatched base-pairs require removal. These are corrected by another repair mechanism, known as mismatch repair.
In this repair mechanism, three gene-products (proteins) are involved — Mut S, Mut L and Mut H. The first step of this repair process consists of binding of the Mut S protein to the mismatched base- pair. The second step involves the recognition of a specific sequence of the template which is -GATC- m E. coli in which A (adenine) is methylated in N-6 position.
The proteins Mut L and Mut H which bind to the unmethylated -GATC- sequence of the new strand form a complex with Mut S which is bound to the mismatch pair. Thereby the mismatch pair is brought close to the -GATC- sequences. The Mut H protein then nicks the unmethylated DNA strand at the GATC site and the mismatch is removed by an exonuclease.
The resulting gap is then filled by DNA polymerase III and DNA ligase (Fig. 9.77):
