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DNA Mismatch Repair-Induced Double-Strand Breaks

Reference: Nowosielska, A. and Marinus, M.G. (2008) DNA Mismatch Repair-Induced Double-Strand Breaks. DNA Repair 7, 48-56.


Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis, that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB(Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O6-meG base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB(Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly.



MutS and MutL Interaction with the Beta Sliding Clamp

Reference: Lopez de Saro FJ, Marinus MG, Modrich P, O'Donnell M. (2006) The beta sliding clamp binds to multiple sites within MutL and MutS. J Biol Chem. 281, 14340-9.

The MutL and MutS proteins are the central components of the DNArepair machinery that corrects mismatches generated byDNA polymerases during synthesis. We find that MutL interacts directly with the beta sliding clamp, a ring-shaped dimeric protein that confers processivity to DNA polymerases by tethering them to their substrates. Interestingly, the interaction of MutL with beta only occurs in the presence of single-stranded DNA. We find that the interaction occurs via a loop in MutL near the ATP-binding site. The binding site of MutL on beta locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with beta without disrupting MutL function with helicase II. In vivo studies reveal that this mutant MutL is no longer functional in mismatch repair. In addition, the human MLH1 has a close match to the proliferating cell nuclear antigen clamp binding motif in the region that corresponds to the beta interaction site in Escherichia coli MutL, and a peptide corresponding to this site binds proliferating cell nuclear antigen. The current report also examines in detail the interaction of . with MutS. We find that two distinct regions of MutS interact with.beta  One is located near the C terminus and the other is close to the N terminus, within the mismatch binding domain. Complementation studies using genes encoding different MutS mutants reveal that the N-terminal  beta interaction motif on MutS is essential for activity n ivivo, but the C-terminal interaction site for . is not. In light of these results, we propose roles for the beta clamp in orchestrating the sequence of events that lead to mismatch repair in the cell.
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The MutS C-Terminus

Reference: Calmann, M.A., Nowosielska, A. and Marinus, M.G. (2005) The MutS C-terminus is essential for mismatch repair activity in vivo. J. Bacteriol. 2005 187: 6577-6579.

An Escherichia coli K-12 strain was constructed with a chromosomal deletion (mutS800) in the mutS gene that produced the removal of the C-terminal 53 amino acids which are not present in the MutS crystal structure. This strain has a MutS null phenotype for mutation avoidance, antirecombination, and sensitivity to cytotoxic agents in a dam mutant background.

MutS Split-Phenotype

Reference: Calmann, M.A., Nowosielska, A. and Marinus, M.G. (2005) Separation of mutation avoidance and antirecombination functions in an Escherichia coli mutS mutant. Nucleic Acids Research 33, 1193-1200.

DNA mismatch repair in Escherichia coli has been shown to be involved in two distinct processes: mutation avoidance, which removes potential mutations arising as replication errors, and antirecombination which prevents recombination between related, but not identical (homeologous), DNA sequences. We show that cells with the mutS800 mutation (which removes the C-terminal 53 amino acids of MutS) on a multicopy plasmid are proficient for mutation avoidance. In interspecies genetic crosses, however, recipients with the mutS800 mutation show increased recombination by up to 280-fold relative to mutS. The MutSD800 protein binds to O6-methylguanine mismatches but not to intrastrand platinated GG cross-links, explaining why dam bacteria with the mutS800 mutation are resistant to cisplatin, but not MNNG, toxicity. The results indicate that the C-terminal end of MutS is necessary for antirecombination and cisplatin sensitization, but less significant for mutation avoidance. The inability of MutS800 to form tetramers may indicate that these are the active form of MutS.


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