A biochemical characterization of the MSH2-MSH6 and MLH1-PMS complexes of Saccharomyces cerevisiae.
Marc Mendillo, Dan Mazur, Richard Kolodner
Ludwig Inst For Cancer Res., UC San Diego, MC 0669, La Jolla, CA, 92093, USA
The MSH2-MSH6 complex is the major mispair recognition complex in the yeast Saccharomyces cerevisiae. Previous studies have shown that in the absence of downstream effector proteins, MSH2-MSH6 dissociates off a mispaired base upon addition of ATP. Alternatively, the MLH1-PMS1 complex, another mismatch repair component, can form a ternary complex with MSH2-MSH6 and DNA in the presence of ATP. Additional studies have implicated the requirement of a free end on DNA in order to observe the ATP induced dissociation of MSH2-MSH6. Here we describe a system for studying end-dependent dissociation using the Lac Repressor-Operator interaction as a method for blocking DNA ends that can be reversed by the addition of isopropylthiogalactoside (IPTG). We find that the MSH2-MSH6 complex undergoes multiple modes of dissociation; one of a rapid, end-dependent nature and a second, relatively slow mode occurring regardless of the state of the DNA ends. Furthermore, upon elimination of the end-dependent mode, there is a shift in equilibrium allowing increased MSH2-MSH6-mispair complex formation at steady-state, which is dependent on the presence of ATP and magnesium. We also show that magnesium, in addition to ATP, is essential in forming an MLH1-PMS1-MSH2-MSH6-DNA ternary complex that is relevant to mismatch repair. We conclude that MLH1-PMS1enhances the stability of MSH2-MSH6 by reducing its rate of end-dependent dissociation observed upon ATP introduction to the MSH2-MSH6-DNA complex.
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