Escherichia coli MutS forms a mispair-dependent ternary complex with MutL that is essential for initiating mismatch repair (MMR) but is structurally uncharacterized, in part owing to its dynamic nature. Here, we used hydrogen/deuterium exchange mass spectrometry and other methods to identify a region in the connector domain (domain II) of MutS that binds MutL and is required for mispairdependent ternary complex formation and MMR. A structurally conserved region in Msh2, the eukaryotic homolog, was required for formation of a mispair-dependent Msh2-Msh6 -Mlh1-Pms1 ternary complex. These data indicate that the connector domain of MutS and Msh2 contains the interface for binding MutL and Mlh1-Pms1, respectively, and support a mechanism whereby mispair and ATP binding induces a conformational change that allows the MutS and Msh2 interfaces to interact with their partners.C ells have evolved a network of DNA repair pathways that respond to various types of genotypic stress to maintain the stability of their genome. For wild-type cells the mutation rate is extremely low (Ϸ1 ϫ 10 Ϫ9 to 1 ϫ 10 Ϫ10 per cell division) (1), which is in part due to DNA mismatch repair (MMR) that removes base-base mismatches and small insertion/deletion mismatches, which arise because of errors in DNA replication, and reduces the error rate of DNA replication by 2 to 3 orders of magnitude (2-5). MMR proteins are also important for recombination and checkpoint responses that lead to the induction of apoptosis in response to some DNA-damaging agents (4, 6, 7). MMR is conserved from bacteria to humans and prevents the development of cancers in humans (8, 9).The initial stages of MMR are similar in both bacteria and eukaryotes. Mispairs in DNA are recognized by the MutS homodimer in Escherichia coli or by one of two heterodimers of MutS homologs, Msh2-Msh6 or Msh2-Msh3, in eukaryotes (2, 10, 11). This complex then recruits the MutL homodimer in E. coli or, in eukaryotes, one of two MutL heterodimeric complexes, Mlh1-Pms1 or Mlh1-Mlh3, in an ATP-dependent manner (12-16). In E. coli, MutS-MutL-DNA ternary complex stimulates the endonucleolytic activity of MutH, which makes single-strand breaks in the unmethylated DNA strand at transiently hemimethylated GATC sites and thus distinguishes the unmethylated daughter DNA strand from the methylated parental DNA strand during and after DNA replication (17-19). The nick serves to mediate excision and strand resynthesis of the newly synthesized DNA to remove the mispair (20)(21)(22). In contrast to E. coli, the downstream events after formation of the ternary complex in eukaryotes, particularly those leading to the initiation of strand-specific MMR, are not well understood.Despite the numerous reports examining the mechanistic features of the MutS-MutL-DNA complex in the initiation of MMR (2) and the available structures of MutS (23,24) and the Nand C-terminal domains of MutL (25, 26), little is known about how MutS interacts with MutL. Recently, mutations in the N-terminal domain of Mlh1 were shown to eli...

We have performed deuterium exchange mass spectrometry (DXMS) to probe the conformational changes that the bacterial MutS homodimer and the homologous eukaryotic heterodimer Msh2-Msh6 undergo when binding to ATP or DNA. The DXMS data support the view that high affinity binding to mispair-containing DNA and low affinity binding to fully base-paired DNA both involve forming rings by MutS protein family dimers around the DNA; however, mispair binding protects additional regions from deuterium exchange. DXMS also reveals two distinct conformations upon binding one or two ATP molecules and that binding of two ATP molecules propagates conformational changes to other regions of the protein complexes. The regions showing major changes in deuterium exchange upon ATP binding tend to occur in regions distinct from those involved in DNA binding, suggesting that although communication occurs between DNA and nucleotide binding, sliding clamps formed by binding both ATP and mispairs could result from the simultaneous action of two independent conformational changes. DNA mismatch repair (MMR)3 recognizes and repairs mispaired nucleotides that arise in DNA as a result of errors during DNA replication, chemical damage to DNA and DNA precursors, and during the formation of heteroduplex recombination intermediates (1-4). The MutS homodimer detects mispairs in DNA in bacteria (2, 5-8), whereas mispaired bases in eukaryotes are recognized by two different partially redundant heterodimers of MutS homologs, Msh2-Msh6 (MutS␣) and Msh2-Msh3 (MutS␤), that have different mispair binding specificities (3, 9 -12). The ability of MutS, as well as Msh2-Msh6 and Msh2-Msh3, to recruit other MMR proteins and trigger downstream events after recognizing mispairs is mediated by dynamic interactions with .Extensive biochemical studies have revealed that the function of MutS and its homologs depends upon extensive communication between the two ATP-binding sites and on communication between the ATP-binding sites and the DNA-binding site, likely mediated by conformational changes that are induced in the protein. Details of these interactions appear to be generally conserved between MutS and its homologs (21, 22), but they are probably best understood in the context of Msh2-Msh6, in which the two ATP-binding sites can be distinguished. In solution, Msh2-Msh6 containing two bound ATP molecules does not form stable complexes with DNA (13); however, under conditions where ATP can be hydrolyzed, the most stable form of the protein contains ADP bound in the Msh2 nucleotide-binding site (23), and this state can bind both base-paired and mispaired DNA, albeit with a higher affinity for mispaired DNA (13, 23). Binding of Msh2-Msh6 to mispaired DNA suppresses ATP hydrolysis (24), primarily at the Msh6 site (23). Thus, mispaired DNA promotes increased steady state levels of ATP-bound Msh6, which facilitates binding of MutL homologs (23,25) and the dissociation of ADP from the Msh2 site (23). Binding of ATP or ATP␥S at the Msh2 site converts the mispair-bound form ...

X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.