A conserved MutS homolog connector domain interface interacts with MutL homologs

Abstract

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...

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

“…Protein complexes analyzed by HDX were introduced to an Orbitrap Elite Mass Spectrometer (ThermoFisher Scientific) for electrospray ionization and accurate mass measurements. DXMS Explorer (Sierra Analytics) was used for the calculation of the deuteration level of all of the peptides as described previously (56,57).…”

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

“…Interactions of hMutS␣ and hMutS␣-hMutL␣ ternary complex with DNA were measured as described with minor modification (33,35). Briefly, 25 nM hMutS␣ was flowed over the DNA in running buffer containing 20 mM Tris-HCl, pH 7.6, 1 mM DTT, 0.005% surfactant P20, 5 mM MgCl 2 , 110 mM KCl at a flow rate of 20 l/min.…”

Dynamic Structural Changes Are Observed upon Collagen and Metal Ion Binding to the Integrin α1 I Domain

“…The DXMS method measures the solvent accessibility of mainchain amides in defined segments of a protein through a combination of time-dependent deuterium exchange, limited proteolysis, and mass spectrometry (33)(34)(35)(36)(37)(38)(39)(40). Percent deuteration is operationally defined as the number of deuterium ions incorporated into a given peptide at a fixed time, divided by the maximum level incorporated at equilibrium.…”

“…Peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) has been used to analyze protein/protein, protein/substrate, protein/inhibitor, protein/solvent, and protein/DNA interactions, as well as protein dynamics and protein conformational changes (33)(34)(35)(36)(37)(38)(39)(40). Here, we utilized DXMS to directly study the solution binding and conformational dynamics of the interaction between ␣1I and full-length type IV collagen.…”