Abstract
DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (KD = 29 nM) while it dramatically decreases above 100 mM (KD>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.
Highlights
MutL homologs (MLH/PMS) are key components of mismatch repair (MMR)
The hidden Markov modeling (HMM) analysis discerned two FRET states resulting from the singlestranded DNA (ssDNA) binding by MutL, which was presented in a transition density plot (Fig. 1C) [18]
The transition density plot represents the transition distribution between two distinct FRET states from 0.44 to 0.26 and from 0.26 to 0.44 along each axis (FRET before transition to FRET after transition). It indicates that the random coiled ssDNA tail that was not bound by MutL displayed a constant FRET efficiency of 0.44
Summary
Mismatch recognition by MutS homologs (MSH) results in long-lived ATP-bound sliding clamps that recruit MLH/ PMS, which in turn stimulate the DNA transaction activities of several downstream effectors. MutL has been suggested to bind ssDNA in the presence of ATP; an activity that may play an important role in its interaction with downstream effectors such as UvrD [3,4,5]. The resulting structure of the ATP-bound MutL appears to form a cavity via a flexible linker that contains a positively charged cleft [9]. This ATP-dependent MLH/ PMS conformational change appears to be modulated by the ATP binding and hydrolysis cycle even in the absence of DNA [11]
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