Single-molecule motions and interactions in live cells reveal target search dynamics in mismatch repair

Abstract

MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches.super-resolution microscopy | DNA replication | bacterial cell biology | Bacillus subtilis | single-cell analysis D NA mismatch repair (MMR) is the highly conserved process responsible for correcting DNA replication errors (1). Although replication errors occur infrequently in bacteria (∼1 error per 31 million bp) (2), the consequences of MMR failure on human health are severe (3). MutS is the first protein involved in the MMR pathway, and it is responsible for detecting rare base-pairing errors. In Bacillus subtilis, MutS then recruits MutL, an endonuclease in most bacteria and eukaryotic organisms, to incise the DNA (4, 5). After MutL incision, the error-containing strand is removed, and the DNA is resynthesized to complete error correction (6).The mechanism by which MutS homologs locate a single mismatch among millions of correctly paired nucleotides has been studied extensively by bulk biochemistry, in vitro atomic force microscopy and single-molecule imaging, and visualizing MutS using in vivo cell biology approaches (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In vitro single-molecule studies largely indicate that MutS operates as a searching clamp diffusing along DNA in a 1D search process (8,10,13). In this model, after mismatch recognition, MutS can dwell at the mismatch before exchanging ADP for ATP, converting into a stable ATP-bound sliding clamp, and subseque...

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

“…Notably, the change from fast to slow motion for PolC at the replisome is similar to the behavior we previously observed for the DNA mismatch repair protein MutS (35). It is possible that both proteins exhibit similar dynamics at the replisome simply because DNA binding on the chromosome occurs more readily at the replication fork where the DNA is unwound and more accessible.…”

MutS2 Promotes Homologous Recombination in Bacillus subtilis

“…Our previous research investigating MutS1 has shown that B. subtilis MutS1-GFP forms foci that are recruited to the site of DNA synthesis (25)(26)(27)(28)48). Further, homologous recombination proteins and nucleotide excision repair proteins have been shown to form foci and localize to the nucleoid, respectively, following MMC treatment (44,49).…”

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

“…As MMR is primarily a coreplicative process (Hombauer et al 2011;Liao et al 2015), most mutations in MMR-deficient cancers are replicative errors. Therefore, the strand biases observed in MSI cancers reveal the biases of corresponding polymerases without the confounding factor of MMR.…”

“…As MMR is primarily a coreplicative process (Hombauer et al 2011;Liao et al 2015), we hypothesized that this asymmetry is due to a joint effect of the differences in rates of mismatches produced by replicative polymerases on the leading and on the lagging strands, and differences in number of mutations repaired by the MMR between the two strands. To investigate this question, we employed data from patients with inherited biallelic MMR deficiency (bMMRD) and somatic mutations in one of the two major replicative polymerases, Pol epsilon (mutated Pol epsilon) or Pol delta (mutated Pol delta).…”