DNA REPAIR

This is only a summary and you will need to add details from Chapter 10 o f Madigan, Martino and Parker, Brock, Biology of Microorganisms, 10th editon. P rentice Hall, publishes. Ionizing radiation causes three types of damage to DNA: Single-strand breaks - mostly sealed by DNA ligase so don't contribute to lethality Double-strand breaks - often lethal because can't be resealed by ligase so degraded by nucleases Alteration of bases - this type of oxidative damage is usually lethal because it forms a replication barrier at that site A UV radiation - 260 nm is wavelength at which maximum absorption occurs for DNA UV - major photoproduct is intrastrand linkage of adjacent pyrimidines, usually thymines, called thymine dimers. Creates distortion in helix and affects replication and transcription. Pol III can't replicate past T-dimer because if puts in A across from dimer, recognizes the weak H-bonding as a mismatch and proofreads. Tries to put in another A, fails. Causes stuttering of Pol III at this site. Block in replication isn't permanent; can have dimer repaired. There are four ways to repair of T dimers in E. coli:

(1) Photoreactivation (aka Light Repair)

phr gene - codes for deoxyribodipyrimidine photolyase that, with cofactor folic acid, binds in dark to T dimer. When light shines on cell, folic acid absorbs the light and uses the energy to break bond of T dimer; photolyase then falls off DNA

(2) Excision Repair (aka Dark Repair)

There are 3 different types of repair mechanisms which use different enzymes but none-the-less follow the same basic principle as outlined in the figure below.

(a) AP Repair (aka Base Excision Repair, BER): Repair of apurinic and apyrimidinic sites on DNA in which base has been removed. Base removed by radiation or DNA glycosylases which sense and remove damaged bases. ung gene codes for uracil-DNA glycosylase which recognizes and removes U in DNA by cleaving the sugar-nitrogen bond to remove the base. AP endonucleases: class I nick at 3' side of AP site and class II nick at 5' side of AP site. Exonuclease removes short region of DNA and DNA Pol I and ligase fill in gap.

(b) UV Damage Repair (also called NER - nucleotide excision repair): NER differs from BER in several ways. It uses different enzymes. Even though there may be only a single "bad" base to correct, its nucleotide is removed along with many other adjacent nucleotides; that is, NER removes a large "patch" around the damage. Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by uvrA, uvrB and uvrC genes). UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction and ligase seals. polA - encodes DNA pol I; mutant was viable (retained normal 5'>3' exo. activity and only 2% of polymerase activity) so Pol I not major replication enzyme, but mutant also had somewhat increased sensitivity to UV so first suggested that played a role in DNA repair. (c) Mismatch Repair (MMR): Accounts for 99% of all repairs Follows behind replication fork. Two ways to correct mistakes made during replication: 1) 3'>5' exonuclease - proofreading 2) Mismatch repair mutH, mutL, mutS and mutU gene products involved (mut for mutator because if gene is mutated, cell has increased levels of spontaneous mutations) How does system recognize progeny strand rather than parent strand as one with mismatch? Because of methylation. DNA methylase (coded for by dam [DNA adenine methylase] locus) methylates 5'-GATC-3' sequence in DNA at A residue. Mismatch from replication recognized by mutL and mutS gene products. mutH gene product nicks DNA strand (progeny strand) on either side of mismatch. DNA helicase II from mutU gene (also called uvrD gene) unwinds DNA duplex and releases nicked region. Gap filled in by DNA Pol I and ligase.

(3) Postreplicative (Recombinational) Translesion Bypass Repair

If T dimer is not repaired, DNA Pol III can't make complementary strand during replication. Postdimer initiation - skips over lesion and leaves large gap (800 bases). Gap may be repaired by enzymes in recombination system - lesion remains but get intact double helix. RecA - coats ssDNA and causes it to invade dsDNA. When stimulated by presence of ssDNA, it also acts as protease to cleave lambda repressor and acts to cause autocatalysis of LexA repressor. recA mutants - very UV-sensitive Now have sister-strand exchange - a type of recombination Translesion bypass. Postreplicative repair is part of SOS response.

(4) SOS Response

UV light is considered be mutagenic - why is this so if all of the UV damage we've talked about is repaired by the 3 repair mechanisms that we have discussed above? In all the three cases, (photoreactivation, excision repair and postreplicative repair) are not mutagenic as the damage is repaired. SOS repair: occurs when cells are overwhelmed by UV damage - this allows the cell to survive but at the cost of mutagenesis. response is only triggered when other repair systems fail as they are overwhelmed by the increased amount of damage so that unrepaired DNA accumulates in the cell. The accumulation of DNA damage leads to repair induction or W-reactivation (Weigle-reactivation). Irradiated lambda phage are more likely to survive in an irradiated rather than. an unirradiated host because SOS system has already been turned on in irradiated host. What triggers the SOS response? Is it the UV-irradiation event or the presence of damaged DNA that triggers the response? If F' cell is UV-irradiated and conjugated to unirradiated F- cell, damaged F' DNA causes induction of repair system (SOS) with maximal enzyme levels in 30 min. SOS regulon includes lexA (autoregulation), recA, uvrA, uvrB, uvrC, umuDC, sulA, sulB, and ssb LexA normally represses about 18 genes sulA and sulB, activated by SOS system, inhibit cell division in order to increase amount of time cell has to repair damage before replication. Each gene has SOS box in promoter. LexA binds SOS box to repress expression. However, LexA catalyses its own breakdown when RecA is stimulated by ssDNA. Non-SOS repair is basically error-free, but SOS repair is error-prone. This is why UV is a mutagen. May be due to RecA binding ssDNA in lesions, which could then bind to DNA Pol III complex passing through this area of the DNA and inhibit 3'>5' exonuclease (proofreading) ability. This makes replication faster but also results in more mutations. This affect on proofreading seems to involve UmuD'-UmuC complex as well. RecA facilitates proteolytic cleavage of UmuD to form UmuD'. The UmuD'-UmuC complex may bind to the RecA-Pol III complex and promote error-prone replication. Also allows Pol III to replicate past a T-dimer but introduces many mutations while doing so Once damage is repaired, RecA no longer catalyzes cleavage of LexA (which is still being made), so uncleaved LexA accumulates and turns the SOS system off.

Why are DNA Repair Systems Necessary? E.coli:

These types of repair, capable of repairing thymine dimers, are so important to bacteria that an E. coli strain that is phr (no photoreactivation), recA (no translesion by pass or SOS), and uvrA (no excision repair) is killed by a single thymine dimer. Xeroderma Pigmentosum (XP): XP is a rare inherited disease of humans which, among other things, predisposes the patient to pigmented lesions on areas of the skin exposed to the sun and an elevated incidence of skin cancer.

It turns out that XP can be caused by mutations in any one of several genes - all of which have roles to play in NER. Some of them: XPA, which encodes a protein that binds the damaged site and helps assemble the other proteins needed for NER. XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD also produce signs of premature aging. [Link] XPF, which cuts the backbone on the 5' side of the damage XPG, which cuts the backbone on the 3' side. Deinococcus radiodurans, a superbug which survives radiation, eats toxic waste: A can of spoiled meat and nuclear waste may appear to have little in common, but the bacterium D. radiodurans thrives in both environments. This bacterium was discovered in 1956 when it was identified as the culprit in a can of spoiled ground beef thought to be radiation sterilized. Scientists subsequently learned that its extreme radiation resistance enables the microbe to survive doses thousands of times higher than would kill most organisms, including humans. Although the ability of the lowly cockroach to withstand radiation has long been admired, it is far surpassed by that of the bacterium D. radiodurans. The remarkable DNA-repair processes of D. radiodurans allow it to stitch together flawlessly its own radiationshattered genome in about 24 hours.

The radiation-resistance profile of D. radiodurans compared to such other organisms as the common intestinal bacterium E. coli, cockroaches, and humans. When older colonies of D. radiodurans are used, their survival extends much farther, to around 17kGy (1.7 mill rads). Scientists believe this extreme radiation resistance may be a side effect of D. radiodurans' ability to survive severe dehydration, whic also fragments DNA. [Nature Biotechnology 18, 85-90 (January 2000)]

Summary for DNA transfer, recomination and repair