Evidence for ATP-dependent Structural Rearrangement of Nuclease Catalytic Site in DNA Mismatch Repair Endonuclease MutL

Abstract

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...

Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL

“…aqMutL, aqMutL CTD, ttMutL, His-tagged ttMutL CTD, and T. thermophilus ␤-clamp were prepared as previously described (19,43,50). Mutants of aqMutL, aqMutL CTD, ttMutL, and Histagged ttMutL CTD were prepared by the same procedure as for wild type proteins.…”

Analysis of the Interaction Interfaces of the N-Terminal Domain from Pseudomonas aeruginosa MutL

“…We hypothesized that NTD could behave differentially since the former have to cope with an additional activity. As mentioned before, ATP binding has been involved in the allosteric control of CTD activity [5], [7], [17], [40]. Taking this into account, we performed all-atom molecular dynamics (MD) simulations of monomeric ATP-bound or ATP-free PaNTD and E. coli LN40 (here on denominated EcNTD).…”

“…This interaction interface is located opposite to the dimerization face, encompassing residues 209–230 and 245–252. Deuterium incorporation assays have previously allowed the detection of a significant ATP-dependent structural rearrangement in the homologue region of the Aquifex aeolicus NTD and that this region may be required for the direct interaction between the NTD and CTD [17]. SBM simulations of PaNTD dimers bound to ADP or ATP indicate that adenine nucleotide binding site is communicated with this putative interaction patch located in residues 208–230.…”

“…NTD nucleotide-dependent conformational changes observed in prokaryotic and eukaryotic MutL homologues, and particularly ATP binding, have been involved in this allosteric control [5], [7], [16]. Recently, a physical NTD-CTD interaction has been demonstrated for Aquifex aeolicus MutL homologue, which possesses endonuclease activity [17]. Therefore, to better understand and elucidate the biochemical and structural regulatory mechanisms underlying CTD endonuclease activity, a deep understanding of the characteristics of NTD from MutL homologues that possess this activity is needed.…”

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…In the case of a ligand-induced conformational change, differences in surface accessibility can be highlighted by HDX while the gain or loss of intramolecular interactions between domains can be monitored by cross-linking [125]. Alternatively, a structural model can be generated based on cross-linking experiments, while deuteration rates can be used to determine the dynamics of the proposed model [126].…”