Abstract
ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces conformational changes in Msh2-Msh6; however, the nature of these conformational changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a conformational change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg(1124). An engineered disulfide bond within this region prevented the ATP-driven conformational change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven conformational change plays a role in mismatch repair.
Highlights
The mismatch recognition complex Msh2-Msh6 undergoes an ATP-mediated conformational change
ATP Binding to Msh2-Msh6 Protects the C Terminus of Msh6 from Trypsin Cleavage—We previously demonstrated that ATP binding protected Msh2-Msh6 from digestion with trypsin (Fig. 1A), consistent with a conformational change [41]
To determine which part of Msh2-Msh6 undergoes the ATP-induced conformational change, bands corresponding to fulllength Msh6 and the 90, 80, and 75-kDa trypsin fragments were excised from a gel, digested to completion with trypsin, and analyzed by mass spectrometry (MS; Fig. 1, B–E)
Summary
The mismatch recognition complex Msh2-Msh undergoes an ATP-mediated conformational change. ATP binding by MutS and its homologs is required for the ability of these proteins to interact with MutL or the eukaryotic MutL homologs, respectively, and thereby mediate downstream events in MMR (18 –20, 24) Together these biochemical results are consistent with the presence of an ATP-induced conformational change in MutS, Msh2-Msh, and Msh2-Msh that propagates from the ATPase domain to other parts of the molecule. Based on structural homology to ATP-bound Rad50 [43], this form of the MutS family of proteins has been called the closed form [41, 42] Consistent with these inferred states, deuterium exchange studies with S. cerevisiae Msh2-Msh demonstrated that ATP binding protects the Msh2-Msh dimer interface between the ATPase domains and exposes the mispair-binding domains [42]. In the studies presented here, we have used a combination of genetic and biochemical approaches to identify a region undergoing an ATP-induced conformational change and demonstrate its functional involvement in MMR
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
