Elsevier

DNA Repair

Volume 20, August 2014, Pages 71-81
DNA Repair

Single molecule studies of DNA mismatch repair

https://doi.org/10.1016/j.dnarep.2014.03.007Get rights and content

Abstract

DNA mismatch repair, which involves is a widely conserved set of proteins, is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair.

Introduction

All organisms require a stable genome. Continuous assault by endogenous and exogenous chemicals and the imperfect fidelities of DNA polymerases tend to degrade the genome. Consequently, multiple DNA repair pathways have evolved to maintain genomic integrity. If these repair pathways fall short, cell cycle checkpoints result that cause cell-cycle arrest and/or apoptosis. These pathways include DNA mismatch repair (MMR), which is responsible for correcting errors made during DNA replication. The MMR proteins also are involved in several other DNA transactions. Inactivation of the MMR genes not only dramatically increases the frequency of mutations, it also decreases apoptosis, increases cell survival, and results in resistance to many chemotherapeutic agents [1], [2], [3]. In humans, mutations in the genes responsible for the initiation of MMR are associated with >80% of hereditary non-polyposis colorectal cancers (HNPCC) and certain sporadic cancers [4]. Patients with cancers associated with defects in MMR genes may be at particular risk because the loss of MMR results in resistance to the cytotoxic effects of several DNA damaging agents, such as cisplatin and alkylating agents, as well as increased mutagenesis due to the inability to repair replication errors generated from copying both normal and damaged bases. Together, these effects are thought to contribute to selective growth advantages for MMR defective cells during multistage carcinogenesis [5]. Understanding the molecular mechanisms that underlie these different activities will be essential for developing effective treatments, with minimal side effects.

MMR is initiated by MutS and MutL homologs, which are highly conserved throughout prokaryotes and eukaryotes. MutS and MutL homologs are dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo [6], [7]. Eukaryotes have multiple heterodimeric MutS and MutL homologs [8], [9], [10], [11], [12], [13], [14]. MSH2-MSH6 (MutSα) is primarily responsible for repairing single base–base mismatches and one and two base insertions or deletions (IDLs), whereas MSH2-MSH3 (MutSβ) is primarily responsible for repairing larger IDLs [11], [15], [16], [17], [18]. MutLα (MLH1-PMS2 in humans, Mlh1-Pms1 in yeast) is the major MutL homolog involved in MMR. The MMR proteins must both locate mismatches in a vast excess of correctly paired DNA and direct repair to the daughter strand.

MMR has been reconstituted in vitro with Escherichia coli and human proteins using plasmid DNA containing a mismatch and a nick (or a hemimethylated GATC site for E. coli) either 5′ or 3′ to the mismatch [19], [20], [21], [22], [23], [24], [25], [26]. In eukaryotes, MMR is initiated by MutSα (or MutSβ) first recognizing a mismatch or IDL (Fig. 1). ATP and mismatch binding induce a conformational change in MutSα, such that it forms a mobile clamp state that can move along the DNA. This activated state of MutSα, in turn, promotes its interaction with one or more MutLα proteins [27], [28], [29], [30], [31], [32], [33], [34], [35]. Subsequently, PCNA, which is a component of the replication apparatus, activates MutLα to incise the daughter strand hundreds of base pairs from a mismatch both distally (preferential) and proximally [36], [37]. Once MutLα nicks the DNA, MutSα activates the exonuclease EXO1 to processively excise the DNA containing the incorrect nucleotide [20], [38], [39], [40]. Alternatively, POLδ/ɛ can initiate strand-displacement synthesis from the nick [41]. Finally, DNA resynthesis is catalyzed by DNA polymerases δ (lagging strand) or ɛ (leading strand), and DNA ligase seals the nick [42], [43], [44]. Because PCNA is loaded asymmetrically on DNA at the replication fork (or at a nick), the PCNA-activated nicking of the DNA by MutLα may serve as a strand-discrimination signal in MMR [45]. For leading strand synthesis, this nicking is thought to be essential to provide a nick 5′ to the mismatch in the daughter strand; however, for lagging strand synthesis, the 5′-end of the Okazaki fragment could also provide a transient strand-discrimination signal [46], [47], [48]. Consistent with this latter suggestion, MutLα is not required for 5′-nick directed repair in vitro [49], [50].

The early steps of MMR are similar in prokaryotes; however, processing of the DNA after mismatch recognition differs in prokaryotes and eukaryotes, which puts different constraints on signaling repair. In prokaryotes, the MutS-MutL initiation complex “directs” UvrD-catalyzed unwinding toward the mismatch, followed by excision by the appropriate exonuclease [6], [7], [51]. In contrast, no helicases are known to be involved in eukaryotic MMR, and EXO1, a double-stranded 5′ to 3′ exonuclease, is the only exonuclease that is clearly involved in repair [7]. Consequently, it is not necessary for MutSα (or MutLα) to confer directionally on EXO1, only to activate it.

Here, we review applications of single molecule experimentation to investigate these MMR phenomena.

Section snippets

MutS searching homoduplex DNA for mismatches

In the 1970s and 1980s, it was proposed that DNA binding proteins underwent 1D diffusion along DNA to explain the observation that the rate of association of proteins with their specific sites on long DNA molecules is faster than theoretically possible based on 3D diffusion alone [52], [53], [54]. Many subsequent biochemical studies, which investigated the DNA length dependence of proteins binding to their specific sites, supported 1D diffusion, but it was the onset of single-molecule tracking

MutS tracks double helix

Single particle tracking studies not only allow the direct observation of proteins diffusing along DNA, but also permit the determination of the relative importance of diffusion with rotation in register with the DNA helix compared to weaker DNA interactions allowing hopping or jumping [60], [63], [64], [66], [69], [70]. By examining the salt dependence of the diffusion rate and/or comparing the observed diffusion coefficients with theoretical predictions of the diffusion coefficients based on

MutS bends DNA during its search

Crystal structures of Taq and E. coli MutS and human MutSα bound to several different mismatched DNA bases and base insertion/deletions [72], [73], [74], [75] reveal only two specific amino acid contacts between MutS or MutSα and the mismatched base: a phenylalanine which stacks with the mismatched base and a glutamate, which forms a hydrogen bond with the N3 of the mismatched thymine or N7 of the mismatched purine [72], [73], [74], [75]. All other interactions between MutS and the DNA are

Conformational properties of MutS-mismatch complexes

Several single-molecule studies have investigated conformational changes in DNA associated with binding of E. coli and Taq MutS to different mismatches. In an early study, AFM was used to directly visualize MutS bound to mismatched and to homoduplex DNA [76]. The capability of AFM to distinguish binding of MutS at homoduplex (nonspecific) DNA sites from binding on mismatches allows identification of conformations unique to the mismatch (or specific site). In addition, analysis of the

Properties of MutL homologs

MutL and MutL homologs are members of the GHL ATPase family [94], [95], [96], [97], [98], which includes DNA Gyrase, Hsp90, Grp94 and the type II topoisomerases. ATP binding and/or hydrolysis induces large conformational changes in GHL proteins, which are thought to be involved in the signaling of cellular processes [94], [96], [99], [100], [101], [102], [103], [104]. Both homodimeric bacterial MutL and heterodimeric eukaryotic MutLα dimerize via their C-terminal domains, which are connected to

Downstream mismatch repair signaling involving MutL

In a recent study [68], Gorman et al. examined the interaction of QD-MutSα and QD-MutLα on λ-DNA containing three GT mismatches spaced by 38 bp, using MutSα and MutLα labeled with QDs with distinct emission wavelengths. Surprisingly, they found that QD-MutLα colocalized with QD-MutSα bound to a mismatch in the presence of ADP with a lifetime of 7.8 min, which is similar to the dwell time of MutSα alone in this assay. This result is in stark contrast to bulk studies, which do not detect any

The future of single molecule methods in studies of DNA mismatch repair

DNA mismatch repair proceeds by a series of transient, multi-protein interactions that present significant challenges to experimentation using traditional biochemical methods. Single molecule approaches are well suited to overcome these challenges and uncover the key signaling events that lead to repair of flawed DNA. In this review, we have described the existing state of the art in applying single molecule investigation to DNA MMR. To date, these studies have mainly focused on the binary

Conflict of interest

The authors, Dorothy Erie and Keith Weninger, declare that they have no conflicts of interest associated with the submitted work.

Acknowledgements

This work was supported by a research scholar grant from the American Cancer Society RSG-10-048 (K.R.W.) and by National Institutes of Health grant GM079480 (D.A.E.) and GM080294 (D.A.E.).

References (127)

  • M. Raschle et al.

    Mutations within the hMLH1 and hPMS2 subunits of the human MutLalpha mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSalpha

    J. Biol. Chem.

    (2002)
  • S. Guo et al.

    Differential requirement for proliferating cell nuclear antigen in 5′ and 3′ nick-directed excision in human mismatch repair

    J. Biol. Chem.

    (2004)
  • I. Iaccarino et al.

    MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2

    Curr. Biol.

    (1996)
  • H. Hombauer et al.

    Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates

    Cell

    (2011)
  • F.A. Kadyrov et al.

    Endonucleolytic function of MutLalpha in human mismatch repair

    Cell

    (2006)
  • M.J. Longley et al.

    DNA polymerase delta is required for human mismatch repair in vitro

    J. Biol. Chem.

    (1997)
  • N. Constantin et al.

    Human mismatch repair: reconstitution of a nick-directed bidirectional reaction

    J. Biol. Chem.

    (2005)
  • Y. Zhang et al.

    Reconstitution of 5′-directed human mismatch repair in a purified system

    Cell

    (2005)
  • A. Pluciennik et al.

    Involvement of the beta clamp in methyl-directed mismatch repair in vitro

    J. Biol. Chem.

    (2009)
  • S.E. Liberti et al.

    Exonuclease 1 preferentially repairs mismatches generated by DNA polymerase alpha

    DNA Repair

    (2013)
  • Y.I. Pavlov et al.

    Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast

    Curr. Biol.

    (2003)
  • J. Genschel et al.

    Mechanism of 5′-directed excision in human mismatch repair

    Mol. Cell

    (2003)
  • J. Genschel et al.

    Functions of MutLalpha, replication protein A (RPA), and HMGB1 in 5′-directed mismatch repair

    J. Biol. Chem.

    (2009)
  • A.D. Riggs et al.

    The lac repressor-operator interaction. 3. Kinetic studies

    J. Mol. Biol.

    (1970)
  • P.H. von Hippel et al.

    Facilitated target location in biological systems

    J. Biol. Chem.

    (1989)
  • C. Bustamante et al.

    Facilitated target location on DNA by individual Escherichia coli RNA polymerase molecules observed with the scanning force microscope operating in liquid

    J. Biol. Chem.

    (1999)
  • M. Guthold et al.

    Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase

    Biophys. J.

    (1999)
  • Y. Harada et al.

    Single-molecule imaging of RNA polymerase–DNA interactions in real time

    Biophys. J.

    (1999)
  • J. Jiang et al.

    Detection of high-affinity and sliding clamp modes for MSH2-MSH6 by single-molecule unzipping force analysis

    Mol. Cell

    (2005)
  • J. Gorman et al.

    Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6

    Mol. Cell

    (2007)
  • J.J. Warren et al.

    Structure of the human MutSalpha DNA lesion recognition complex

    Mol. Cell

    (2007)
  • I. Tessmer et al.

    Mechanism of MutS searching for DNA mismatches and signaling repair

    J. Biol. Chem.

    (2008)
  • M.J. Schofield et al.

    Interaction of Escherichia coli MutS and MutL at a DNA mismatch

    J. Biol. Chem.

    (2001)
  • Y. Yang et al.

    Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy

    Methods

    (2003)
  • M.L. Mendillo et al.

    Analysis of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 complexes with DNA using a reversible DNA end-blocking system

    J. Biol. Chem.

    (2005)
  • C. Ban et al.

    Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair

    Cell

    (1999)
  • C. Ban et al.

    Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis

    Cell

    (1998)
  • R. Dutta et al.

    GHKL, an emergent ATPase/kinase superfamily

    Trends Biochem. Sci.

    (2000)
  • X. Hu et al.

    Monovalent cation dependence and preference of GHKL ATPases and kinases

    FEBS Lett.

    (2003)
  • A. Fedier et al.

    Mutations in DNA mismatch repair genes: implications for DNA damage signaling and drug sensitivity

    Int. J. Oncol.

    (2004)
  • P. Modrich et al.

    Mismatch repair in replication fidelity, genetic recombination, and cancer biology

    Annu. Rev. Biochem.

    (1996)
  • R. Fishel

    The selection for mismatch repair defects in hereditary nonpolyposis colorectal cancer: revising the mutator hypothesis

    Cancer Res.

    (2001)
  • T.A. Kunkel et al.

    DNA mismatch reapir

    Annu. Rev. Biochem.

    (2005)
  • R.R. Iyer et al.

    DNA mismatch repair: functions and mechanisms

    Chem. Rev.

    (2006)
  • S. Santucci-Darmanin et al.

    Homologs of MutS and MutL during mammalian meiosis

    Med. Sci. (Paris)

    (2003)
  • H. Flores-Rozas et al.

    The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
  • B.D. Harfe et al.

    DNA mismatch repair and genetic instability

    Annu. Rev. Genet.

    (2000)
  • S.M. Lipkin et al.

    MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability

    Nat. Genet.

    (2000)
  • S.M. Lipkin et al.

    Meiotic arrest and aneuploidy in MLH3-deficient mice

    Nat. Genet.

    (2002)
  • M.J. Schofield et al.

    DNA mismatch repair: molecular mechanisms and biological function

    Annu. Rev. Microbiol.

    (2003)
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