Single molecule studies of DNA mismatch 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.).
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2019, ChemosphereCitation Excerpt :Colocalization of HSP 70 and nucleotide excision repair (NER) proteins was observed in the nuclei of human bronchial epithelial cells treated with the genotoxic agent benzo(a)pyrene (BaP) (Yang et al., 2009) and enhancement of NER by HSP 70 was evidenced by the faster removal of Bap-induced DNA adducts in human cells transfected with a plasmid expressing HSP 70 (Duan et al., 2014). DNA mismatch repair (MMR) maintains genome integrity by correcting potentially mutagenic simple base mispairs and loops of extra bases that generated during DNA replication and recombination (Erie and Weninger, 2014; Groothuizen and Sixma, 2016). Eukaryotic MMR is a nick-directed process initiated by the binding of heterodimeric protein complexes composed of E. coli MutS homologs (MSH) to mismatched nucleotides (Erie and Weninger, 2014; Groothuizen and Sixma, 2016).