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In this article we will discuss about the five main DNA repair mechanisms that are studied in eukaryotes. The mechanisms are: 1. Photo-Reactivation or Photo-Repair 2. Dark or Excision Repair 3. Nucleotide Excision Repair 4. Mismatch Repair 5. Post-Replication Repair.
Mechanism # 1. Photo-Reactivation or Photo-Repair:
This mechanism was discovered when bacteria that had been heavily irradiated with UV rays were found to have recovered their colony forming ability when stored in the presence of visible light. UV rays are a major source of damage to DNA and have been studied extensively for repair of DNA lesions (pyrimidine dimers).
UV induces joining of adjacent pyrimidines on the same DNA strand by the formation of a cyclobutane ring resulting from saturation of the double bonds between carbons 5 and 6. The presence of dimers in DNA blocks transcription or replication at the site of damage.
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One method of repairing UV-induced dimmers involves reversal of dimerisation reaction. The process is called photo-reactivation. It uses energy of visible light to break the ring structure.
Thus the original pyrimidine bases remain in DNA but are restored to their normal state of individual pyrimidines. Repair of UV-induced damage by photo-reactivation is common in prokaryotes and eukaryotes. A peculiar feature is that, photo-reactivation may not be present in all tissues of an organism.
In the crab Gecarcinus it is absent in the midgut gland but present in most other tissues. In chickens it is present in the fibroblasts and brain, but is absent in liver, kidney, skeletal muscle and some other tissues. Many species of animals as well as humans do not have this mechanism of DNA repair.
Mechanism # 2. Dark or Excision Repair:
Many bacteria can repair UV induced damage during storage in the dark. There are some mutant strains of E. coli which are extremely sensitive to UV. The bacterium Micrococcus radiodurans is highly resistant.
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After UV irradiation both sensitive and resistant bacteria show similar numbers of pyrimidine dimers in their DNA indicating differences in their repair mechanisms. When irradiated bacteria are incubated, the resistant strains show excision and release of pyrimidine dimers whereas sensitive strains do not.
It is now known that the process of excision repair involves several enzymatic steps: cutting of a single strand near the dimer; removal or excision of several bases including a dimer; synthesis of a new single strand complementary to the one opposite the excised dimer and linking the new strand to the original one (Fig. 20.7).
The excision repair mechanism is also effective on other types of damage produced by UV, X and gamma radiation and on bases modified by alkylating agents in eukaryotic organisms.
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Xeroderma pigmentosum (XP) is an autosomal recessive hereditary skin disease of man much studied for excision repair. Affected persons develop skin cancer when exposed to sunlight. When cultured cells from XP patients are irradiated with UV, they are found to be more sensitive than normal cells.
The sensitivity has been associated with defective excision repair, XP cells showing 10-70% repair as compared to normal cells. Later studies by Cleaver (1969) and others showed that XP cells were deficient in the initial stage of excision repair, which involves production of single strand breaks by endonucleolytic enzyme.
Normal cells could produce large number of single strand breaks which later disappeared. XP cells produced no such breaks. Thus pyrimidine dimers are not lost from DNA of irradiated XP cells.
Mechanism # 3. Nucleotide Excision Repair:
The repair of damage by removal of dimers by excision, described above, is also called nucleotide excision repair because the damaged bases, the pyrimidine dimer, are removed as part of a stretch of oligonucleotides. In E.coli, nucleotide excision repair involves protein products of three genes, uvrA, uvrB, uvrC. The protein uvrA recognises damaged DNA and recruits uvrB and uvrC to the damaged site.
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Uvrb and uvrC then cleave on the 3′ and 5′ sides of the damaged site, respectively, so as to excise an oligonucleotide consisting of 12 or 13 bases. The uvrABC complex is called the excinuclease owing to its ability to excise an oligonucleotide. A helicase enzyme then removes the damage-containing oligonucleotide from the DNA molecule, the resulting gap is filled by DNA polymerase I and joined by ligase.
Nucleotide excision repair system has been found in eukaryotes, and studied extensively in yeasts and humans. In yeasts several genes for DNA repair, called RAD (radiation sensitivity) genes have been identified.
In humans, DNA repair genes have been described through studies on patients with Xeroderma pigmentosum described above. At least seven different repair genes, XPA , XPB, …. XPG that are mutated in patients with Xeroderma pigmentosum. The enzymes encoded by these genes have been identified.
Mechanism # 4. Mismatch Repair:
A third excision repair system recognises mismatched bases that are incorporated during DNA replication. Many of the mismatched bases are removed by the proofreading activity of DNA polymerase. The remaining ones are corrected by the mismatch repair system which scans newly replicated DNA.
Mechanism # 5. Post-Replication Repair:
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In a strain of E. coli which was defective for excision of pyrimidine dimers from its DNA, it was found that the length of the newly synthesised DNA was equal to the distance between pyrimidine dimers.
Obviously DNA replication is inhibited in regions where pyrimidine dimers are present. But if the cells are incubated subsequently the average length of newly synthesised DNA increases until it is similar to that found in un-irradiated cells.
The mechanism for repair is not well understood. Perhaps there are several processes involved in restoring normal replication between pyrimidine dimers, and the resulting gaps are subsequently joined. There is some information on post-replication repair in eukaryotes.
If mouse and Chinese hamster cells (which have very little excision repair) receive UV irradiation in two fractions, cell survival is greater than if the whole dose is given in one fraction. This is true only if the cells pass through the S phase between the two fractions. It appears therefore that the repair process and the S phase are associated.
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It has been shown by several workers that the essential features of post replication repair in mammals are synthesis of new DNA strands in S phase leaving extensive gaps which are eventually closed.
Since pyrimidine dimers inhibit DNA synthesis, the number of gaps corresponds to the number of pyrimidine dimers. The size of the gaps is approximately 800-1000 nucleotides long. The process of filling the gaps is inhibited by hydroxyurea, caffeine and theophylline.
In E. coli post replication repair is associated with genes at the rec loci namely rec A, rec B and rec C. A mutation in any of these loci results in loss of ability to integrate donor cell DNA by recombination. These loci are also sensitive to the effects of UV radiation.
