Analysis of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 complexes with DNA using a reversible DNA end-blocking system.
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.
Mendillo
Marc L
ML
Ludwig Institute for Cancer Research, Department of Medicine and Cancer Center, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA.
Mazur
Dan J
DJ
Kolodner
Richard D
RD
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020137:Base Pair Mismatch; D020029:Base Pairing; D015374:Biosensing Techniques; D002021:Buffers; D002352:Carrier Proteins / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D047468:Immunoprecipitation; D008274:Magnesium / Q000737:chemistry; D008957:Models, Genetic; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D002021:Buffers; D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; C098790:MSH6 protein, S cerevisiae; C088858:PMS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; C489751:MSH2 protein, S cerevisiae; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D008274:Magnesium