Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair

Abstract

The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/ MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion-and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA. P ostreplicative mismatch repair (MMR) corrects errors in DNA synthesis before they lead to genomic instability (1-3). MMR increases the fidelity of DNA replication up to 1,000-fold, and MMR defects in humans cause hereditary nonpolyposis colorectal cancer and may influence the onset of other tumors (1). MutSα and MutLα are conserved eukaryotic protein complexes necessary for MMR. MutSα is responsible for recognition of mismatches and small insertion/deletion loops (1-3), whereas MutLα harbors an endonuclease activity necessary for cleavage of the lesion-bearing DNA strand (4, 5).The challenges faced during MMR can be illustrated by considering that Saccharomyces cerevisiae should incur only approximately two mismatches per cell cycle (6). MutSα must find these rare lesions, MutLα must search for lesion-bound MutSα, and the lesion-bound MutSα/MutLα complex must search the flanking DNA for signals that distinguish the parental and daughter strands (1-3). Models describing how DNA-binding proteins search for specific targets include 3D diffusion (i.e., jumping), 1D hopping, 1D sliding, and intersegmental transfer; the latter three are categorized as facilitated diffusion because they allow target association rates exceeding limits imposed by 3D diffusion (7-10). New single-molecule and NMR techniques have led to resurgent interest in understanding how proteins locate targets (11-13), and using single-molecule imaging we previously demonstrated that MutSα and MutLα can undergo facilitated diffusion on undamaged DNA through 1Dsliding and 1D hopping, respectively (14, 15). However, no single-molecule study has directly revealed proteins searching for and subsequently engaging a target site through 1D diffusion (i.e., 1D sliding or 1D hopping) (7), and the inability to visualize target capture also prevents investigation of questions regarding downstream MMR events.Here we used nano...

Mismatch repair protein hMSH2–hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate

“…In the presence of ADP and ATP, the k on2 were 2.5-and 30-fold faster than the k on for the HsRAD51-bound short synthetic D-loop obtained under identical conditions (Table 1 and Table S3). These results support previous studies that demonstrated that MSH proteins search and then bind to a DNA mismatch through a 2D sliding mechanism (14,(17)(18)(19)(20). Taken as a whole, we conclude that hMSH2-hMSH6 recognizes mismatch within D-loop formed by RAD51 and is capable of forming an ATP-bound sliding clamp.…”

“…ATP binding by hMSH2-hMSH6 results in the formation of a sliding clamp that is released from the mismatch and diffuses along the DNA duplex (17,21). In the case of HR intermediates, the ATP-bound hMSH2-hMSH6 sliding clamp is confined within the mismatch-containing D-loop until it comes in contact with displaced ssDNA strand or with the branch points at the end of the D-loop (Figs.…”

“…The structure of an ATP-bound MSH dimer has yet to be solved. However, numerous biochemical and single-molecule studies have demonstrated that mismatch recognition triggers ADP→ATP nucleotide exchange, which results in a conformational transition that converts the MSH dimer into an ATP-bound stable sliding clamp that disengages from the mismatch and freely diffuses on the adjacent dsDNA for 8-10 min (14)(15)(16)(17)(18)(19)(20)(21). The formation of an ATP-bound MSH sliding clamp is followed by recruitment of MutL homologs (MLH/PMS) (22,23).…”

“…The formation of an ATP-bound MSH sliding clamp is followed by recruitment of MutL homologs (MLH/PMS) (22,23). With the exception of Gram-negative enteric bacteria such as Escherichia coli, the MLH/PMS of most species including the human hMLH1-hPMS2 contain an endogenous endonuclease activity required for processing the error-containing DNA strand (17,24).…”

Dynamic control of strand excision during human DNA mismatch repair

“…For comparison, the cellular concentration of HsMSH2-HsMSH6 has been estimated to be ∼250 nM (24). All of the HsMSH2-HsMSH6 proteins appeared to diffuse randomly on the mismatched DNA consistent with ATPbound sliding clamps as described previously (20,(25)(26)(27).…”