DNA Repair

Methyl-directed mismatch repair (MMR)

In all organisms genomic integrity is normally maintained by a variety of DNA repair pathways, including MMR and nucleotide excision repair (NER).61 Secondary DNA structures formed during DNA synthesis, especially within single-stranded regions containing repetitive tracts, may be hazardous for genome stability if not removed by repair activities. MMR pathway is a fundamental system involved in maintaining genomic integrity because in addition to correcting mismatched base pairs, it also repairs some nonclassical DNA structures such as small hairpins and unpaired regions within DNA. Upon inactivation of MMR increased heterogeneity is observed at simple repetitive DNA (e.g., mono- and dinucleotides) in bacteria, yeast and mammals. The associations of defective MMR and an elevated genetic instability at simple DNA repeats are particularly strong for hereditary nonpolyposis cancer.

The investigations of a role of methyl-directed mismatch repair in TRS instability were an important step in studying the molecular mechanisms leading to the accumulation of dynamic mutations among triplet repeats. Notably, studies performed in bacteria and yeast have identified that MMR had contrasting effects on the genetic stability of TRS.62-64 Although instability of the TRS is linked to human disorders, functional similarities between the MMR in prokaryotes, lower eukaryotes and humans justified this study. For example, E.coli strains with defective MMR had a reduced occurrence of large deletions (more than 8 repeats) from plasmids harboring long CTG/CAG. By contrast, mutations in MMR proteins increased the frequency of small length changes (less than 8 repeats) in shorter CTG/CAG repeats in E.coli and S. cerevisiae.

To clarify these apparently conflicting results, Parniewski et al have used a variety of lengths of CTG/CAG tracts (ranging from 25 to 175 units) to determine the effects of MMR on repeat tract stability in E.coli.65 They showed that depending on the length of repeats the functional MMR proteins act to promote large deletions (usually more than 8 repeats) in CTG/CAG tracts, but significantly prevent length changes (both, expansions and deletions) of less than 8 repeats. Not only the length of the TRS influenced the incidence of deletions in CTG/CAG but also the instability was dependent on the purity of TRS (i.e., presence of interruptions) as well as the cell growth conditions. One plausible explanation of this distinctive behavior of the MMR proteins acting on the TRS is the propensity of the triplet repeats to undergo different structural transitions depending on length of the repeated motif. Since short TRS are more likely to form slipped structures as opposed to the long ones, which will rather tend to assemble into stable hairpins therefore, different local Non-B-DNA structures may trigger particular cellular mechanisms. Considering this and results from other groups, we propose a model which links structural properties of the (CTG)n to the polymerase pausing and bypass synthesis within DNA tracts being repaired by the MMR (Fig.1.5). Following the DNA slippage of the complementary strands in double-stranded TRS region, small loops are formed on both strands and therefore are recognized by functional MMR proteins. The repair process leads to the excision of large segments of non-methylated strand spanning a region containing loopouts and to the formation of single-stranded regions on the complementary strand. Short single-stranded TRS tracts (of less than approx. 100 units) are much less prone to form stable hairpins than the long ones and resynthesis of the complementary strand will result in neither deleted nor expanded TRS. In the absence of functional MMR proteins, the same tract will be subjected to SILC pathway and consequently small expansions and deletions will gradually accumulate within the repeated motif after subsequent generations of cells. Therefore, repair of small loops that could arise on relatively short CTG/CAG tracts would stabilize the TRS and lack of this repair function will have an opposite effect. Conversely, the same repair pathway acts differently on long tracts ofthe TRS. Following the formation of slipped-stranded structures, recognition of small loops and excision of one DNA strand by the MMR protein complex will result in long single-stranded stretches in the CTG region which will self-pair and form stable hairpins. If during resynthesis of a gap DNA

Figure 6. The possible pathways of the NER-generated genetic instability of long transcribed CTG/CAG tracts in orientation II. The CTG hairpin formed on the lagging strand template during TRS replication (shaded circle) may be removed by the UvrA dimer (left panel) and once the correct template for the repair synthesis is restored, the TRS is replicated with no length changes. Bypass synthesis in NER deficient strains will result in large deletions (middle panel). In the absence of the functional UvrA protein the UvrBC complex may specifically recognize and excise the CTG hairpin, which would also lead to large deletions.

Figure 6. The possible pathways of the NER-generated genetic instability of long transcribed CTG/CAG tracts in orientation II. The CTG hairpin formed on the lagging strand template during TRS replication (shaded circle) may be removed by the UvrA dimer (left panel) and once the correct template for the repair synthesis is restored, the TRS is replicated with no length changes. Bypass synthesis in NER deficient strains will result in large deletions (middle panel). In the absence of the functional UvrA protein the UvrBC complex may specifically recognize and excise the CTG hairpin, which would also lead to large deletions.

Figure 7. Recombination pathways of Double-strand break repair. (Frame) Double-strand breaks initiate nearly all homologous recombination pathways and are the start point for the 5'^3' exonucleolytic digestion which leaves 3' ends of donor DNA duplex free to invade the template molecule. (A) Szostak et al. model.75 Invasion leads to the displacement of a template strand (D-loop formation) [i]. Free 3' ends of the donor molecule are the priming sites for the DNA synthesis followed by the formation of ā€˛Holliday junctions". Resolution of these structures occurs by cutting each junction in one of two directions (open and closed arrowheads) [ii]. Cutting both junctions in the same orientation results in gene conversion while the opposite cuts yield gene conversion associated with crossover [iii]. (B) Synthesis-Dependent Strand Annealing (SDSA) model. After invasion and DNA synthesis donor strands unwind from the template and reanneal without crossed-over product formation [iii]. (C) Alternative, Bubble Migration SDSA model. Only one 3' ended donor strand invades template molecule [ii]. DNA synthesis occurs within migrating bubble formed by the displaced strand [iii]. After unwinding from the template donor strands containing repeated sequences (multiple arrowheads) may reanneal in out-of-frame order what may lead to expansions or deletions [iv]. Several modifications of SDSA as well as other recombination pathways may be involved in repeated sequences instability (for a review see ref. 74).

Note that in case of the repeated sequences, polymerase slippage and idling may occur during repair synthesis, which may additionally increase the rate of expansions.

polymerase bypasses the hairpin the "repaired" molecule would contain big deletions. However, when the CAG strand serves as a template for repair synthesis (inverse orientation of CTG/CAG tract) the nascent DNA would be able to produce stable hairpins, which would possibly, cause DNA polymerase to stall. Further multiple polymerase slippages, the relocation of newly synthesized repeated DNA fragment and idling synthesis will result in large expansions of the TRS.

Our model presented above explains opposite results concerning the role of the MMR system in generating TRS instabilities in bacteria and yeast, obtained in different laboratories. However, how MMR affects the frequency of expansion events in humans remains unclear. Moreover, long CAG/CTG repeats from the gene associated with Huntington's disease in humans were shown to be less prone to expand in transgenic mice with defective MSH2 protein.66 Together, these in vivo observations suggest that mutations in MMR enzymes are not required for expansions of TRS in mammals, and the involvement of this repair system in TRS related diseases needs to be more extensively studied.

Nucleotide excision repair (NER)

Nucleotide excision repair is another major cellular defense system in both prokaryotes and eukaryotes. This pathway efficiently recognizes and repairs a vast majority of damages, including bulky DNA adducts and DNA cross-links that cause significant distortion of the helix, as well as less distortive lesions such as methylated bases. Also, the involvement of NER in the repair of DNA loops in vitro has been reported.67,68 In humans, defects in NER proteins cause at least three hereditary disorders, including xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.61

Since unusual DNA structures can form in some TRS in vivo and may therefore invoke destabilization of double-stranded helix, they are also likely to trigger the NER proteins and during the repair process enhance repeat tract instability. Studies in E.coli revealed that bacterial NER proteins influence the genetic stability of the TRS in a complex manner.69 First of all, the stability, as demonstrated by previous investigations was highly dependent on the length of the repeated tract and the orientation of TRS insert relative to the origin of replication. The instability was only observed for long CTG/CAG tracts (175 units) in orientation II, where the CTG strand served as a template for lagging strand synthesis. However, in long-term (multigenerational) growth of the wild type strain and its isogenic uvrA or uvrB mutants, the rate of deletions in strain lacking functional UvrA protein was significantly higher as compared to strain that lacked only UvrB. In E.coli UvrA is required for damage recognition. The affinity of the UvrA protein to single-stranded DNA, specifically to bubbles and loops may be responsible for the recognition and binding to the CTG hairpins in their single-stranded loop region.67,70 Binding of the UvrA to unusual conformations may destabilize such structures allowing the correct copying of the entire repeat. Others have demonstrated that absence of the single-stranded-DNA-binding protein (SSB) in vivo similarly led to an increased frequency of large deletions within the triplet repeats.71 Very high stability of long CTG/CAG tracts in strains lacking functional UvrB suggests that this protein may be involved in processing of unusual structures within repeats and allows deletions to occur. In some in vitro studies specific recognition and excision of bubbles within double-stranded DNA by the UvrBC endonucleolytic complex was demonstrated.72 An alternative scenario is that in the absence of the UvrA protein, the CTG hairpin may be also a substrate for the cellular endonucleolytic activities. Such nicked DNA may be degraded in vivo which would similarly lead to deletions. The possible pathways of the CTG hairpins processing by NER are presented on Figure 1.6.

Interestingly, the genetic differences in the stability of long CTG/CAG tracts between uvrA and uvrB mutants were apparent only if the TRS were transcribed. Transcription through the TRS may additionally stabilize CTG hairpins by introducing negative supercoils behind RNA polymerase complex. It is important to note that the NER pathway is well suited to repair transcribed strands. Any kind of RNA polymerase pausing triggers transcription-repair coupling factor (TRCF). This protein attracts NER components to the transcribed region providing prompt removal of DNA lesions. One might assume that formation of the hairpin structures on the template strand as well as on the nascent RNA may lead to RNA polymerase stalling. Napierala et al demonstrated that CUG repeats form extremely stable, length-dependent, self-complementary structures. This strongly supports the hypothesis that structural aberrations within the TRS are causative for their genetic instability.

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