DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.
DNA MMR (mismatch repair) is an excision repair system that removes mismatched bases generated primarily by failure of the 3'-5' proofreading activity associated with replicative DNA polymerases. MutL proteins homologous to human PMS2 are the endonucleases that introduce the entry point of the excision reaction. Deficiency in PMS2 function is one of the major etiologies of hereditary non-polyposis colorectal cancers in humans. Although recent studies revealed that the CTD (C-terminal domain) of MutL harbours weak endonuclease activity, the regulatory mechanism of this activity remains unknown. In this paper, we characterize in detail the CTD and NTD (N-terminal domain) of aqMutL (Aquifex aeolicus MutL). On the one hand, CTD existed as a dimer in solution and showed weak DNA-binding and Mn2+-dependent endonuclease activities. On the other hand, NTD was monomeric and exhibited a relatively strong DNA-binding activity. It was also clarified that NTD promotes the endonuclease activity of CTD. NTD-mediated activation of CTD was abolished by depletion of the zinc-ion from the reaction mixture or by the substitution of the zinc-binding cysteine residue in CTD with an alanine. On the basis of these results, we propose a model for the intramolecular regulatory mechanism of MutL endonuclease activity.
DNA mismatch repair (MMR) greatly contributes to genome integrity via the correction of mismatched bases that are mainly generated by replication errors. Postreplicative MMR excises a relatively long tract of error-containing single-stranded DNA. MutL is a widely conserved nicking endonuclease that directs the excision reaction to the error-containing strand of the duplex by specifically nicking the daughter strand. Because MutL apparently exhibits nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of MutL has been argued. Recent studies suggest ATP-dependent conformational and functional changes of MutL, indicating that the regulatory mechanism involves the ATP binding and hydrolysis cycle. In this study, we investigated the effect of ATP binding on the structure of MutL. First, a cross-linking experiment confirmed that the N-terminal ATPase domain physically interacts with the C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass spectrometry clarified that the binding of ATP to the N-terminal domain induces local structural changes at the catalytic sites of MutL C-terminal domain. Finally, on the basis of the results of the hydrogen/deuterium exchange experiment, we successfully identified novel regions essential for the endonuclease activity of MutL. The results clearly show that ATP modulates the nicking endonuclease activity of MutL via structural rearrangements of the catalytic site. In addition, several Lynch syndrome-related mutations in human MutL homolog are located in the position corresponding to the newly identified catalytic region. Our data contribute toward understanding the relationship between mutations in MutL homolog and human disease.The DNA mismatch repair (MMR) 4 system greatly contributes to the replication fidelity of DNA by correcting the mismatched bases generated by the errors of DNA polymerases (1-3). The early reactions of eukaryotic MMR are performed mainly by MutS␣ (MSH2/MSH6 heterodimer) and MutL␣ (PMS2/MLH1 and PMS1/MLH1 heterodimers in Homo sapiens and Saccharomyces cerevisiae, respectively). It is reported that hereditary dysfunctions of these proteins are a major cause of Lynch syndrome (also known as hereditary non-polyposis colorectal cancer) in humans (4,5). MutS␣ recognizes the mismatched bases and stimulates MutL␣, which has a latent nicking endonuclease activity that introduces the entry or termination point of the subsequent excision reaction (6 -8). MutL␣ directs MMR to the daughter strand by specifically nicking the discontinuous strand in cooperation with replication protein A as well as the loaded form of proliferating cell nuclear antigen in the presence of ATP (7). After the error-containing strand is excised by EXO1, DNA polymerase ␦ resynthesizes the strand to complete the repair reaction.The MMR systems of the majority of bacteria except Escherichia coli and other ␥-proteobacteria are believed to resemble the eukaryotic system (8). The majority of bacteria possess MutS and MutL dimers whose fundamental characterist...
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