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Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs. Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9)-mediated genome editing offers a powerful approach as a potential therapy for monogenic human genetic diseases. Precise template-free base deletions can be achieved through microhomology-mediated end joining (MMEJ) repair and depend on local target site sequence. The DNA repair pathway choice in CRISPR-Cas9 induced-double-strand breaks (DSBs) is regulated by several key factors including the cell cycle, target site sequence and chromatin structure, and the identity of the donor DNA template. Homology-directed repair (HDR)-related DNA repair pathways in response to CRISPR-Cas9 induced-DSBs in mammalian cells are complicated and relatively inefficient. Different DNA repair pathways might be used to repair each end at a DSB resulting in the potential for asymmetric repair. [ABSTRACT FROM AUTHOR]

Abstract Homologous recombination (HR) is a universally conserved mechanism used to maintain genomic integrity. In eukaryotes, HR is used to repair the spontaneous double strand breaks (DSBs) that arise during mitotic growth, and the programmed DSBs that form during meiosis. The mechanisms that govern mitotic and meiotic HR share many similarities, however, there are also several key differences, which reflect the unique attributes of each process. For instance, even though many of the proteins involved in mitotic and meiotic HR are the same, DNA target specificity is not: mitotic DSBs are repaired primarily using the sister chromatid as a template, whereas meiotic DBSs are repaired primarily through targeting of the homologous chromosome. These changes in template specificity are induced by expression of meiosis-specific HR proteins, down-regulation of mitotic HR proteins, and the formation of meiosis-specific chromosomal structures. Here, we compare and contrast the biochemical properties of key recombination intermediates formed during the pre-synapsis phase of mitotic and meiotic HR. Throughout, we try to highlight unanswered questions that will shape our understanding of how homologous recombination contributes to human cancer biology and sexual reproduction. [ABSTRACT FROM AUTHOR]

Srs2 is a superfamily 1 (SF1) helicase and antirecombinase that is required for genome integrity. However, the mechanisms that regulate Srs2 remain poorly understood. Here, we visualize Srs2 as it acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. We demonstrate that Srs2 is a processive translocase capable of stripping thousands of Rad51 molecules from ssDNA at a rate of ∼50 monomers/s. We show that Srs2 is recruited to RPA clusters embedded between Rad51 filaments and that multimeric arrays of Srs2 assemble during translocation on ssDNA through a mechanism involving iterative Srs2 loading events at sites cleared of Rad51. We also demonstrate that Srs2 acts on heteroduplex DNA joints through two alternative pathways, both of which result in rapid disruption of the heteroduplex intermediate. On the basis of these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates. [ABSTRACT FROM AUTHOR]

Summary Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. [ABSTRACT FROM AUTHOR]

Summary Shelterin is a six-subunit protein complex that plays crucial roles in telomere length regulation, protection, and maintenance. Although several shelterin subunits have been studied in vitro, the biochemical properties of the fully assembled shelterin complex are not well defined. Here, we characterize shelterin using ensemble biochemical methods, electron microscopy, and single-molecule imaging to determine how shelterin recognizes and assembles onto telomeric repeats. We show that shelterin complexes can exist in solution and primarily locate telomeric DNA through a three-dimensional diffusive search. Shelterin can diffuse along non-telomeric DNA but is impeded by nucleosomes, arguing against extensive one-dimensional diffusion as a viable assembly mechanism. Our work supports a model in which individual shelterin complexes rapidly bind to telomeric repeats as independent functional units, which do not alter the DNA-binding mode of neighboring complexes but, rather, occupy telomeric DNA in a “beads on a string” configuration. [ABSTRACT FROM AUTHOR]

Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. [ABSTRACT FROM AUTHOR]

Summary Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin. [ABSTRACT FROM AUTHOR]

MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA. We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA. We demonstrate that polymer assembly is initiated by a stochastic nucleation event. After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB. MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes. In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes. Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA. Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA. Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation. This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior. [ABSTRACT FROM AUTHOR]

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (-350 nucleotides per second) in the 5'-30 direction over relatively long distances (-29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA. [ABSTRACT FROM AUTHOR]

Homologous recombination is an important DNA repair pathway that plays key roles in maintaining genome stability. Escherichia coli RecA is an ATP-dependent DNA-binding protein that catalyzes the DNA strand exchange reactions in homologous recombination. RecA assembles into long helical filaments on single-stranded DNA, and these presynaptic complexes are responsible for locating and pairing with a homologous duplex DNA. Recent single molecule studies have provided new insights into RecA behavior, but the potential influence of ATP in the reactions remains poorly understood. Here we examine how ATP influences the ability of the RecA presynaptic complex to interact with homologous dsDNA. We demonstrate that over short time regimes, RecA presynaptic complexes sample heterologous dsDNA similarly in the presence of either ATP or ATPγS, suggesting that initial interactions do not depend on ATP hydrolysis. In addition, RecA stabilizes pairing intermediates in three-base steps, and stepping energetics is seemingly unaltered in the presence of ATP. However, the overall dissociation rate of these paired intermediates with ATP is ~4-fold higher than with ATPγS. These experiments suggest that ATP plays an unanticipated role in promoting the turnover of captured duplexDNAintermediates as RecA attempts to align homologous sequences during the early stages of recombination. [ABSTRACT FROM AUTHOR]

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively. [ABSTRACT FROM AUTHOR]

During meiosis, the two DNA recombinases Rad51 and Dmc1 form specialized presynaptic filaments that are adapted for performing recombination between homologous chromosomes. There is currently a limited understanding of how these two recombinases are organized within the meiotic presynaptic filament. Here, we used single molecule imaging to examine the properties of presynaptic complexes composed of both Rad51 and Dmc1. We demonstrate that Rad51 and Dmc1 have an intrinsic ability to self-segregate, even in the absence of any other recombination accessory proteins. Moreover, we found that the presence of Dmc1 stabilizes the adjacent Rad51 filaments, suggesting that cross-talk between these two recombinases may affect their biochemical properties. Based upon these findings, we describe a model for the organization of Rad51 and Dmc1 within the meiotic presynaptic complex, which is also consistent with in vivo observations, genetic findings, and biochemical expectations. This model argues against the existence of extensively intermixed filaments, and we propose that Rad51 and Dmc1 have intrinsic capacities to form spatially distinct filaments, suggesting that additional recombination cofactors are not required to segregate the Rad51 and Dmc1 filaments. [ABSTRACT FROM AUTHOR]

Rad52 is a highly conserved protein involved in the repair of DNA damage. Human RAD52 has been shown to mediate single-stranded DNA (ssDNA) and is synthetic lethal with mutations in other key recombination proteins. For this study, we used single-molecule imaging and ssDNA curtains to examine the binding interactions of human RAD52 with replication protein A (RPA)-coated ssDNA, and we monitored the fate of RAD52 during assembly of the presynaptic complex. We show that RAD52 binds tightly to the RPA-ssDNA complex and imparts an inhibitory effect on RPA turnover. We also found that during presynaptic complex assembly, most of the RPA and RAD52 was displaced from the ssDNA, but some RAD52-RPA-ssDNA complexes persisted as interspersed clusters surrounded by RAD51 filaments. Once assembled, the presence of RAD51 restricted formation of new RAD52-binding events, but additional RAD52 could bind once RAD51 dissociated from the ssDNA. Together, these results provide new insights into the behavior and dynamics of human RAD52 during presynaptic complex assembly and disassembly. [ABSTRACT FROM AUTHOR]

Homologous recombination plays key roles in double-strand break repair, rescue, and repair of stalled replication forks and meiosis. The broadly conserved Rad51/RecA family of recombinases catalyzes the DNA strand invasion reaction that takes place during homologous recombination. We have established single-stranded (ss)DNA curtain assays for measuring individual base triplet steps during the early stages of strand invasion. Here, we examined how base triplet stepping by RecA, Rad51, and Dmc1 is affected by DNA sequence imperfections, such as single and multiple mismatches, abasic sites, and single nucleotide insertions. Our work reveals features of base triplet stepping that are conserved among these three phylogenetic lineages of the Rad51/RecA family and also reveals lineage-specific behaviors reflecting properties that are unique to each recombinase. These findings suggest that Dmc1 is tolerant of single mismatches, multiple mismatches, and even abasic sites, whereas RecA and Rad51 are not. Interestingly, the presence of single nucleotide insertion abolishes recognition of an adjacent base triplet by all three recombinases. On the basis of these findings, we describe models for how sequence imperfections may affect base triplet recognition by Rad51/RecA family members, and we discuss how these models and our results may relate to the different biological roles of RecA, Rad51, and Dmc1. [ABSTRACT FROM AUTHOR]

Abstract: Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54–Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54–Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1. [Copyright &y& Elsevier]

Saccharomyces cerevisiae RDH54 is a key member of the evolutionarily conserved RAD52 epistasis group of genes needed for homologous recombination and DNA double strand break repair. The RDH54-encoded protein possesses a DNA translocase activity and functions together with the Rad51 recombinase in the D-loop reaction. By chromatin immunoprecipitation (ChIP), we show that Rdh54 is recruited, in a manner that is dependent on Rad51 and Rad52, to a site-specific DNA double strand break induced by the HO endonuclease. Because of its relatedness to Swi2/Snf2 chromatin remodelers, we have asked whether highly purified RdhS4 possesses chromatin-remodeling activity. Importantly, our results show that Rdh54 can mobilize a mononucleosome along DNA and render nucleosomal DNA accessible to a restriction enzyme, indicative of a chromatin-remodeling function. Moreover, Rdh54 co-operates with Rad5l in the utilization of naked or chromatinized DNA as template for D-loop formation. We also provide evidence for a strict dependence of the chromatin-remodeling attributes of Rdh54 on its ATPase activity and N-terminal domain. Interestingly, an N-terminal deletion mutant (rdh54Δ102) is unable to promote Rad51-mediated D-loop formation with a chromatinized template, while retaining substantial activity with naked DNA. These features of Rdh54 suggest a role of this protein factor in chromatin rearrangement during DNA recombination and repair. [ABSTRACT FROM AUTHOR]