Analysis of the Interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 Complexes with DNA Using a Reversible DNA End-blocking System

Abstract

The Lac repressor-operator interaction was used as a reversible DNA end-blocking system in conjunction with an IAsys biosensor instrument (Thermo Affinity Sensors), which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of mismatch repair proteins to move along the DNA. The MSH2-MSH6 complex bound to a mispaired base was found to be converted by ATP binding to a form that showed rapid sliding along the DNA and dissociation via the DNA ends and also showed slow, direct dissociation from the DNA. In contrast, the MSH2-MSH6 complex bound to a base pair containing DNA only showed direct dissociation from the DNA. The MLH1-PMS1 complex formed both mispair-dependent and mispair-independent ternary complexes with the MSH2-MSH6 complex on DNA. The mispair-independent ternary complexes were formed most efficiently on DNA molecules with free ends under conditions where ATP hydrolysis did not occur, and only exhibited direct dissociation from the DNA. The mispair-dependent ternary complexes were formed in the highest yield on DNA molecules with blocked ends, required ATP and magnesium for formation, and showed both dissociation via the DNA ends and direct dissociation from the DNA.Errors that occur during DNA replication result in base-base mismatches and small insertion/deletion mismatches that if left uncorrected are fixed in the DNA as mutations by subsequent rounds of DNA replication. The DNA mismatch repair (MMR) 1 system normally corrects such errors in the cell and is highly conserved from bacteria to humans (1-5). Defects in the system lead to increased rates of accumulation of mutations, and in humans, inherited and somatic defects in MMR result in increased development of cancer (6 -9). The mechanism of MMR is best understood in Escherichia coli (3)(4)(5). In E. coli the MutS protein, which appears to function as a homodimer, serves as the mispair recognition factor (10 -13). MutL, another homodimer, interacts with the MutS complex in the presence of a mispair and ATP and activates the endonucleolytic activity of MutH (11, 14 -20). MutH makes single strand breaks in the newly synthesized daughter strand at transiently unmethylated GATC sequences, allowing the unwinding of the DNA by UvrD coupled with the degradation of the error-containing strand by one of at least four redundant exonucleases (21, 22). Then DNA polymerase III holoenzyme can resynthesize the DNA strand leaving a nick that is subsequently sealed by DNA ligase (23). In eukaryotic cells, three different MutS homologs form two different heterodimeric complexes, called MSH2-MSH6 (MutS␣) and MSH2-MSH3 (MutS␤) that together recognize base-base mispairs and insertion/deletion loops (1, 2, 24 -35). Similarly, three different MutL homologs form two different heterodimeric complexes, called MLH1-PMS1 (MutL␣; PMS1 Saccharomyces cerevisiae is PMS2 in humans) and MLH1-MLH3, that function in MMR (36 -38). Like their bacterial homologs, the MSH and MLH complexe...

A conserved MutS homolog connector domain interface interacts with MutL homologs

“…2A). MutL did not bind to DNA in the absence of MutS, consistent with previous studies with MutL and the eukaryotic homolog Mlh1-Pms1 (13,14). MutS-205 had a modest defect in MutL binding, whereas MutS-211 ( Fig.…”

“…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)(13)(14)(15)(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)(18)(19).…”

“…First, the MutS-MutL-DNA ternary complex and the eukaryotic MSH-MLH-DNA complex are dynamic and exhibit rapid dissociation from DNA (2,12,13,19,29), which may explain the failure of most large-scale physical interaction studies to identify the Saccharomyces cerevisiae ternary complexes (30)(31)(32)(33)(34). Second, both ATP binding and mispair binding are required for the interaction of MutS with MutL and Msh2-Msh6 with Mlh1-Pms1 (2,12,13,19). These cofactors likely transiently induce conformational changes required for ternary complex formation (2).…”

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

“…Several reports in the literature indicate that upon binding ATP, MutS/Msh proteins appear to release the mismatch by sliding away from the site [15,36]. But, ATP binding to MutS/Msh also facilitates formation of ternary complexes containing MutS and MutL proteins and the mismatch [12,15,16].…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…While movement of the clamp was originally proposed to occur via an ATP-hydrolysis driven model or a diffusion-driven model, subsequent work supports the latter. ATP-induced dissociation of yMsh2-Msh6 from a mismatch occurs preferentially from DNA ends though direct dissociation from the DNA is also observed Mendillo et al, 2005). Finally, although MutS and MutL (and their eukaryotic homologues) have been observed to form quasi-stable complexes at sites of mismatches in vitro (Blackwell et al, 2001;Räschle et al, 2002;Schofield et al, 2001 b), yeast and human complexes have also been observed to move along the DNA contour in the presence of ATP in surface plasmon resonance experiments (Blackwell et al, 2001;Mendillo et al, 2005).…”