Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

Abstract

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.

Mismatch binding, ADP–ATP exchange and intramolecular signaling during mismatch repair

“…1) [25,28-30]. Recent AFM and sm-FRET studies of Taq and E. coli MutS have revealed formation of a slightly bent/unbent DNA complex, which is also considered an important intermediate during the process of recognition [15,24,31-33]. …”

“…Mismatch type, sequence context, even the orientation of MutS binding to an asymmetric mismatch (e.g., Phe stacking against T versus G in G:T) can affect repair efficiency [14,33-37]. These variables likely affect the conformational dynamics of the DNA and protein and hence the lifetime of the final mismatch recognition complex, which appears to be a critical factor for subsequent signaling of repair (e.g., yeast MutSα dissociates about 100-fold faster from the poorly repaired 2-Aminopurine:T mismatch than from the best repaired G:T mismatch) [38].…”

Is Thymidine Glycol Containing DNA a Substrate of E. coli DNA Mismatch Repair System?

“…Fluorescence intensity ( I ) and anisotropy ( r ) were measured using Tecan Infinite F200 fluorescence plate reader (Switzerland). Three filter sets were used, namely Green (λ ex  = 475 nm, λ em  = 525 nm), Red (λ ex  = 575 nm, λ em  = 625 nm), and FRET (λ ex  = 475 nm, λ em  = 625 nm) [35]. The anisotropy values for the maximal binding of MutS with the duplexes V–X were estimated (Figure S5 in File S1).…”

Asymmetric perturbations of signalling oligomers

“…An elegant example of this is a study of the homodimeric multidrug efflux protein EmrE, where bulk and single-molecule FRET measurements were used as additional evidence for an asymmetric antiparallel arrangement of the two subunits (Morrison et al, 2012). Single-molecule FRET in general has proven to be a highly valuable biophysical tool for distinguishing between asymmetric and symmetric protein complexes (Takei et al, 2012) and revealing directionality in macromolecular interactions (Cristóvao et al, 2012). Additional single-molecule optical techniques such as fluorescent-molecule video imaging have been used in recent years with great success to obtain a dynamic insight into receptor oligomerization and mobility on the surface of intact cells (Kasai and Kusumi, 2014).…”