ABSTRACT
G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
Subject(s)
DNA Primase , DNA Replication , DNA-Binding Proteins , G-Quadruplexes , Genomic Instability , MutS Homolog 2 Protein , Transcription Factors , Humans , Transcription Factors/metabolism , Transcription Factors/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , MutS Homolog 2 Protein/metabolism , MutS Homolog 2 Protein/genetics , DNA Primase/metabolism , DNA Primase/genetics , Telomere Homeostasis , DNA Damage , HEK293 Cells , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , DNA-Directed DNA PolymeraseABSTRACT
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Genome , DNA/genetics , Optical TweezersABSTRACT
Geometric morphometrics is widely used to quantify morphological variation between biological specimens, but the fundamental influence of operator bias on data reproducibility is rarely considered, particularly in studies using photographs of live animals taken under field conditions. We examined this using four independent operators that applied an identical landmarking scheme to replicate photographs of 291 live Atlantic salmon (Salmo salar L.) from two rivers. Using repeated measures tests, we found significant inter-operator differences in mean body shape, suggesting that the operators introduced a systematic error despite following the same landmarking scheme. No significant differences were detected when the landmarking process was repeated by the same operator on a random subset of photographs. Importantly, in spite of significant operator bias, small but statistically significant morphological differences between fish from the two rivers were found consistently by all operators. Pairwise tests of angles of vectors of shape change showed that these between-river differences in body shape were analogous across operator datasets, suggesting a general reproducibility of findings obtained by geometric morphometric studies. In contrast, merging landmark data when fish from each river are digitised by different operators had a significant impact on downstream analyses, highlighting an intrinsic risk of bias. Overall, we show that, even when significant inter-operator error is introduced during digitisation, following an identical landmarking scheme can identify morphological differences between populations. This study indicates that operators digitising at least a sub-set of all data groups of interest may be an effective way of mitigating inter-operator error and potentially enabling data sharing.
Subject(s)
Information Dissemination , Salmo salar , Animals , Reproducibility of Results , Research Design , RiversABSTRACT
Resolution of Holliday junctions is a critical intermediate step of homologous recombination in which junctions are processed by junction-resolving endonucleases. Although binding and cleavage are well understood, the question remains how the enzymes locate their substrate within long duplex DNA. Here we track fluorescent dimers of endonuclease I on DNA, presenting the complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We show that the enzyme binds remotely to dsDNA and then undergoes 1D diffusion. Upon encountering a four-way junction, a catalytically-impaired mutant remains bound at that point. An active enzyme, however, cleaves the junction after a few seconds. Quantitative analysis provides a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is probably applicable to many junction resolving enzymes.
Subject(s)
DNA, Cruciform , DNA , DNA/metabolism , Deoxyribonuclease I/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Holliday Junction Resolvases/metabolism , Nucleic Acid ConformationABSTRACT
The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.
Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing/methods , Genetic Therapy/methods , Single Molecule ImagingABSTRACT
There are strong signals that the selection forces favouring the expression of long-distance sea migration by Atlantic salmon (Salmo salar) are changing. Unlike many other behavioural traits, the costs of migration are incurred before any fitness benefits become apparent to the migrant. The expression of this behaviour has thus been shaped by selection forces over multiple generations and cannot respond to short interval (within a single generation) environmental change as many other behavioural traits can. Here we provide a framework to examine the evolutionary and ecological consequences of a sustained increase in migration cost. We argue that Atlantic salmon may have entered an evolutionary trap, where long-distance sea migration has become maladaptive because of shifting environmental conditions. We predict that if higher migration costs (affecting survivorship and ultimately fitness) persist, then shifting selection pressures will result in continuing declines in population size. We suggest, however, that in some populations there is demonstrable capacity for evolutionary rescue responses within the species which is to be found in the variation in the expression of migration. Under a scenario of low to moderate change in the selection forces that previously promoted migration, we argue that disruptive, sex-based selection would result in partial migration, where females retain sea migration but with anadromy loss predominantly in males. With more acute selection forces, anadromy may be strongly selected against, under these conditions both sexes may become freshwater resident. We suggest that as the migration costs appear to be higher in catchments with standing waters, then this outcome is more likely in such systems. We also speculate that as a result of the genetic structuring in this species, not all populations may have the capacity to respond adequately to change. The consequences of this for the species and its management are discussed.
Subject(s)
Salmo salar , Animals , Biological Evolution , Female , Male , Phenotype , Population Density , Pressure , Salmo salar/geneticsABSTRACT
DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3' to 5' polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.
Subject(s)
DNA Breaks, Double-Stranded , DNA Helicases , DNA Repair , Rad51 Recombinase , Replication Protein A , DNA , DNA Helicases/metabolism , DNA, Single-Stranded , Rad51 Recombinase/metabolism , Replication Protein A/metabolismABSTRACT
Using optical tweezers, we investigate target search and cleavage by CRISPR-Cas12a on force-stretched λ-DNA. Cas12a uses fast, one-dimensional hopping to locate its target. Binding and cleavage occur rapidly and specifically at low forces (≤5 pN), with a 1.8 nm rate-limiting conformational change. Mechanical distortion slows diffusion, increases off-target binding but hinders cleavage.
Subject(s)
Bacterial Proteins/chemistry , CRISPR-Associated Proteins/chemistry , DNA/chemistry , Endodeoxyribonucleases/chemistry , CRISPR-Cas Systems , Models, Molecular , Optical TweezersABSTRACT
Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force-extended duplex DNA in a two-step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono-intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the "light-switch" properties of this complex on binding DNA, it can be readily used as a long-lived luminescent label for duplex or negatively supercoiled DNA through a unique "load-and-lock" protocol.
Subject(s)
Coordination Complexes/chemistry , DNA Probes/chemistry , DNA/analysis , Ruthenium/chemistry , Molecular StructureABSTRACT
Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).
Subject(s)
DNA, Single-Stranded/chemistry , DNA/chemistry , Single Molecule Imaging/methods , Green Fluorescent Proteins/chemistry , Humans , Optical Imaging , Optical Tweezers , Rad51 Recombinase/chemistry , Recombinant Proteins/chemistry , Replication Protein A/chemistryABSTRACT
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Carrier Proteins/genetics , DNA, Helminth/genetics , DNA-Binding Proteins/genetics , Rad51 Recombinase/genetics , Recombinational DNA Repair , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/metabolism , DNA Breaks, Double-Stranded , DNA, Helminth/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Protein Binding , Rad51 Recombinase/metabolism , Replication Protein A/genetics , Replication Protein A/metabolism , Signal Transduction , Single Molecule ImagingABSTRACT
Fishways are commonly employed to improve river connectivity for fishes, but the extent to which they cater for natural phenotypic diversity has been insufficiently addressed. We measured differential upstream passage success of three wild brown trout (Salmo trutta) phenotypes (anadromous, freshwater-resident adult and parr-marked), encompassing a range of sizes and both sexes, at a Larinier superactive baffle fishway adjacent to a flow-gauging weir, using PIT telemetry (n = 160) and radio telemetry (n = 53, double tagged with PIT tags). Fish were captured and tagged downstream of the weir in the autumn pre-spawning period, 2017, in a tributary of the River Wear, England, where over 95% of tributary spawning habitat was available upstream of the weir. Of 57 trout that approached the weir-fishway complex, freshwater-resident adult and parr-marked phenotypes were less successful in passing than anadromous trout (25%, 36%, and 63% passage efficiency, respectively). Seventy-one percent of anadromous trout that passed upstream traversed the weir directly. Although the fishway facilitated upstream passage, it was poor in attracting fish of all phenotypes (overall attraction efficiency, 22.8%). A higher proportion (68.2%) of parr-marked trout that approached the weir were male and included sexually mature individuals, compared with that of freshwater-resident (37.8%) and anadromous trout (37.0%). The greater passage success of anadromous trout was likely due to their greater size and locomotory performance compared to the other phenotypes. Barriers and fishways can act as selection filters, likely the case in this study, and greater consideration needs to be given to supporting natural diversity in populations when proposing fishway designs to mitigate river connectivity problems.
Subject(s)
Rivers , Trout , Animals , Ecosystem , England , Female , Male , PhenotypeABSTRACT
The purpose of this investigation was to quantify the association between 5 vs. 5 small sided games (SSG) running performance and physiological performance during the Yo-YoIR1 test to ascertain the utility of SSGs as a potential fitness test modality within elite professional soccer players. Twenty-three (n = 23) elite male professional soccer players (mean ± SD age 25.3 ± 3.1 yrs, mass: 76 ± 9 kg, height: 176 ± 9 cm) were assessed. Players completed an intermittent aerobic fitness test (Yo-YoIR1) and a 5 vs. 5 SSGs protocol for the purpose of the study. During all SSGs players wore GPS (Statsports 10-Hz, Viper Pod, Newry, Northern Ireland) and HR monitors (Polar, Oy Kemple, Finland) with these measures related to Yo-YoIR1 running performance. Results revealed SSGs running performance (TD; m) and physiological performance (HR) showed the lowest CV% (< 5%), with high speed movements, accelerations and decelerations highlighting higher CV% during SSGs. Possibly small to possibly very large associations were observed for running performance during 5 vs. 5 SSGs and Yo-YoIR1 performance, with negative associations observed between physiological performance during SSG and YoYoIR1 running performance. To conclude, the current study observed how running performance during a standardised 5 vs. 5 SSG protocol within elite soccer cohorts is associated with the Yo-YoIR1 running performance. Given the low CV%, repeatability and large association of global running performance and internal load measures during a 5 vs. 5 SSG with Yo-YoIR1 performance, this particular soccer specific SSG protocol potentially supplements traditional non-sport specific testing assessments.
ABSTRACT
Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a "permanent bridge" resisting forces over 80 pN and a force-sensitive "reversible bridge." The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. "Permanent" cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.
Subject(s)
Adenosine Triphosphate/chemistry , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA, Fungal/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Adenosine Triphosphate/metabolism , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA, Fungal/metabolism , Humans , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
The nematode Caenorhabditis elegans has been central to the understanding of metazoan biology. However, C. elegans is but one species among millions and the significance of this important model organism will only be fully revealed if it is placed in a rich evolutionary context. Global sampling efforts have led to the discovery of over 50 putative species from the genus Caenorhabditis, many of which await formal species description. Here, we present species descriptions for 10 new Caenorhabditis species. We also present draft genome sequences for nine of these new species, along with a transcriptome assembly for one. We exploit these whole-genome data to reconstruct the Caenorhabditis phylogeny and use this phylogenetic tree to dissect the evolution of morphology in the genus. We reveal extensive variation in genome size and investigate the molecular processes that underlie this variation. We show unexpected complexity in the evolutionary history of key developmental pathway genes. These new species and the associated genomic resources will be essential in our attempts to understand the evolutionary origins of the C. elegans model.
ABSTRACT
CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
Subject(s)
Bacteriophage lambda/genetics , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , DNA, Viral/metabolism , DNA Cleavage , DNA, Viral/genetics , Escherichia coli/virology , Fluorescence Resonance Energy Transfer , Gene Editing , Microfluidics , Microscopy, Confocal , Optical Tweezers , RNA, Guide, Kinetoplastida/genetics , Streptococcus pyogenes/enzymologyABSTRACT
Acoustic telemetry is an important tool for studying the behaviour of aquatic organisms in the wild.VEMCO high residence (HR) tags and receivers are a recent introduction in the field of acoustic telemetry and can be paired with existing algorithms (e.g. VEMCO positioning system [VPS]) to obtain high-resolution two-dimensional positioning data.Here, we present results of the first documented field test of a VPS composed of HR receivers (hereafter, HR-VPS). We performed a series of stationary and moving trials with HR tags (mean HR transmission period = 1.5 s) to evaluate the precision, accuracy and temporal capabilities of this positioning technology. In addition, we present a sample of data obtained for five European perch Perca fluviatilis implanted with HR tags (mean HR transmission period = 4 s) to illustrate how this technology can estimate the fine-scale behaviour of aquatic animals.Accuracy and precision estimates (median [5th-95th percentile]) of HR-VPS positions for all stationary trials were 5.6 m (4.2-10.8 m) and 0.1 m (0.02-0.07 m), respectively, and depended on the location of tags within the receiver array. In moving tests, tracks generated by HR-VPS closely mimicked those produced by a handheld GPS held over the tag, but these differed in location by an average of ≈9 m.We found that estimates of animal speed and distance travelled for perch declined when positional data for acoustically tagged perch were thinned to mimic longer transmission periods. These data also revealed a trade-off between capturing real nonlinear animal movements and the inclusion of positioning error.Our results suggested that HR-VPS can provide more representative estimates of movement metrics and offer an advancement for studying fine-scale movements of aquatic organisms, but high-precision survey techniques may be needed to test these systems.
ABSTRACT
The CRISPR-Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA-RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.
Subject(s)
Bacterial Proteins/chemistry , DNA/chemistry , Endonucleases/chemistry , RNA, Guide, Kinetoplastida/chemistry , RNA/chemistry , Bacterial Proteins/metabolism , Biocatalysis , CRISPR-Associated Protein 9 , Clustered Regularly Interspaced Short Palindromic Repeats , DNA/genetics , Endonucleases/metabolism , Nucleic Acid Conformation , RNA/genetics , RNA, Guide, Kinetoplastida/geneticsABSTRACT
Nursing home (NH) health information technology (IT) is becoming more prevalent across the country. Currently, a national sample of NHs is being surveyed for 3 consecutive years to determine trends in NH IT sophistication (e.g., measures of IT capabilities, extent of IT use, IT integration with internal and external stakeholders). IT sophistication is measured in resident care, clinical support, and administrative activities. The current article provides details of the differences in NH IT sophistication reported by administrators completing Year 1 and Year 2 surveys. IT in clinical support (i.e., laboratory, pharmacy, and radiology) had the greatest differences. This difference is expected because these areas typically require external contracts, making it dificult to fit IT with existing workflows, which is important for sustained adoption. [Journal of Gerontological Nursing, 43(1), 17-21.].
Subject(s)
Information Technology , Nursing Homes/organization & administration , Organizational Innovation , Surveys and QuestionnairesABSTRACT
Cells use homology-dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology-based mechanisms involves nuclease-dependent DNA end resection, which generates long tracts of single-stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re-synthesis of resected DNA We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re-synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single-stranded gap and terminate further resection.