Search

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3′ non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1–Rad10 is the structure-specific endonuclease that cleaves the tails in 3′ non-homologous tail removal (3′ NHTR). Rad1–Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1–Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2–Msh3 and Saw1 recruit Rad1–Rad10 in 3′ NHTR; Rad14 recruits Rad1–Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3′ NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3′ NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A–Rad10 to recombination intermediates is defective. Interactions among rad1R218A–Rad10 and Msh2–Msh3 and Saw1 are altered and rad1R218A–Rad10 interactions with RPA are compromised. We propose a model in which Rad1–Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1–Rad10 and Msh2–Msh3, Saw1 and Msh2–Msh3 and Rad1–Rad10 and RPA. When any of these interactions is altered, 3′ NHTR is impaired.

Yeast Rad1–Rad10 (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1–Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1–Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1–Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1–Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1–Rad10 removes ICL via NER, whereas in S/G2, Rad1–Rad10 facilitates NER-independent replication-coupled ICLR., The yeast Rad1–Rad10 complex has multiple roles in DNA damage repair. Here the authors uncover mutants that uncouple the roles in UV excision repair and non-NER functions.

Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.

Summary DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.

Homologous recombination (HR) is a universally conserved and accurate DNA double strand break (DSB) repair pathway. In humans, HR is initiated by the Mre11/Rad50/Nbs1 (MRN) complex, which rapidly localized to the DSB and recruits nucleases that process the free DNA ends for downstream recombination. This process must occur on chromatin, but little is known about the first steps of HR on a nucleosome coated DNA. Here, we use high-throughput DNA curtains to visualize the first steps of human HR. We show that MRN scans DNA via one-dimensional facilitated diffusion. Remarkably, MRN uses its many DNA-binding modes to bypass nucleosomes and other roadblocks as it searches for DSBs. Next, MRN recruits Exonuclease 1 (Exo1), which uses its 5’→3’ nuclease activity to process the free DNA ends. Exo1 is a processive enzyme that can digest thousands of base pairs of DNA in a single resection event. However, RPA and other single-stranded DNA binding proteins (SSBs) inhibit Exo1 by stripping the nuclease from DNA. RPA inhibition is not species-specific, and requires at least three of its many DNA-binding domains. Strikingly, SOSS1—a recently identified mammalian SSB that is required for DSB repair in human cells—supports long-range Exo1 nuclease activity. Our results provide an integrated model for how DSB repair is initiated on a crowded DNA track and how single-stranded DNA-binding proteins regulate the first steps of human DSB repair.

Single-molecule studies of protein–DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains—a method for organizing arrays of DNA molecules on a fluid lipid bilayer—has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900 000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3—a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments.