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Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex.

ABSTRACT: Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.

SUBMITTER: Crickard JB

PROVIDER: S-EPMC7560196 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Project description:Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

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Rdh54/Tid1 inhibits Rad51-Rad54-mediated D-loop formation and limits D-loop length.

Project description:Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

| S-EPMC7695457 | biostudies-literature

In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation.

Project description:Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.

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New Phosphorylation Sites of Rad51 by c-Met Modulates Presynaptic Filament Stability.

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| S-EPMC6468871 | biostudies-literature

Schizosaccharomyces pombe Rdh54 (TID1) acts with Rhp54 (RAD54) to repair meiotic double-strand breaks.

Project description:We report the characterization of rdh54+, the second fission yeast Schizosaccharomyces pombe Rad54 homolog. rdh54+ shares sequence and functional homology to budding yeast RDH54/TID1. Rdh54p is present during meiosis with appropriate timing for a meiotic recombination factor. It interacts with Rhp51 and the meiotic Rhp51 homolog Dmc1 in yeast two-hybrid assays. Deletion of rdh54+ has no effect on DNA damage repair during the haploid vegetative cell cycle. In meiosis, however, rdh54Delta shows decreased spore viability and homologous recombination with a concomitant increase in sister chromatid exchange. The rdh54Delta single mutant repairs meiotic breaks with similar timing to wild type, suggesting redundancy of meiotic recombination factors. Consistent with this, the rdh54Delta rhp54Delta double mutant fails to repair meiotic double strand breaks. Live cell analysis shows that rdh54Delta rhp54Delta asci do not arrest, but undergo both meiotic divisions with near normal timing, suggesting that failure to repair double strand breaks in S. pombe meiosis does not result in checkpoint arrest.

| S-EPMC266785 | biostudies-literature

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51-DNA filaments.

Project description:Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.

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Bridge-induced chromosome translocation in yeast relies upon a Rad54/Rdh54-dependent, Pol32-independent pathway.

Project description:While in mammalian cells the genetic determinism of chromosomal translocation remains unclear, the yeast Saccharomyces cerevisiae has become an ideal model system to generate ad hoc translocations and analyze their cellular and molecular outcome. A linear DNA cassette carrying a selectable marker flanked by perfect homologies to two chromosomes triggers a bridge-induced translocation (BIT) in budding yeast, with variable efficiency. A postulated two-step process to produce BIT translocants is based on the cooperation between the Homologous Recombination System (HRS) and Break-Induced Replication (BIR); however, a clear indication of the molecular factors underlying the genetic mechanism is still missing. In this work we provide evidence that BIT translocation is elicited by the Rad54 helicase and completed by a Pol32-independent replication pathway. Our results demonstrate also that Rdh54 is involved in the stability of the translocants, suggesting a mitotic role in chromosome pairing and segregation. Moreover, when RAD54 is over-expressed, an ensemble of secondary rearrangements between repeated DNA tracts arise after the initial translocation event, leading to severe aneuploidy with loss of genetic material, which prompts the identification of fragile sites within the yeast genome.

| S-EPMC3629078 | biostudies-literature

Terminal association of Rad54 protein with the Rad51-dsDNA filament.

Project description:Rad54 protein is a Snf2-related dsDNA-specific ATPase essential for homologous recombination mediated by Rad51 protein, the eukaryotic RecA ortholog. Snf2-related enzymes couple ATP hydrolysis with translocation on dsDNA to remodel or dissociate a wide variety of protein-dsDNA complexes. Rad54 and Rad51 interact through species-specific contacts and mutually stimulate their biochemical activities. Specifically, Rad51 bound to dsDNA, the product of homologous recombination after DNA-strand exchange, stimulates the Rad54 ATPase up to 6-fold, leading to the turnover of Rad51 in the product complex. Electron microscopy visualized the Rad51-Rad54 interaction on dsDNA, showing that an oligomeric form of Rad54 associates preferentially with termini of the Rad51-dsDNA filament. Our data support a mechanism of processive dsDNA-Rad51 filament dissociation by the translocating Rad54 protein.

| S-EPMC1502528 | biostudies-literature

Human telomeric proteins occupy selective interstitial sites.

Project description:Human telomeres are bound and protected by protein complexes assembled around the six core telomeric proteins RAP1, TRF1, TRF2, TIN2, TPP1, and POT1. The function of these proteins on telomeres has been studied extensively. Recently, increasing evidence has suggested possible roles for these proteins outside of telomeres. However, the non-canonical (extra-telomeric) function of human telomeric proteins remains poorly understood. To this end, we systematically investigated the binding sites of telomeric proteins along human chromosomes, by performing whole-genome chromatin immunoprecipitation (ChIP) for RAP1 and TRF2. ChIP sequencing (ChIP-seq) revealed that RAP1 and TRF2 could be found on a small number of interstitial sites, including regions that are proximal to genes. Some of these binding sites contain short telomere repeats, suggesting that telomeric proteins could directly bind to interstitial sites. Interestingly, only a small fraction of the available interstitial telomere repeat-containing regions were occupied by RAP1 and TRF2. Ectopically expressed TRF2 was able to occupy additional interstitial telomere repeat sites, suggesting that protein concentration may dictate the selective targeting of telomeric proteins to interstitial sites. Reducing RAP1 and TRF2 expression by RNA interference led to altered transcription of RAP1- and TRF2-targeted genes. Our results indicate that human telomeric proteins could occupy a limited number of interstitial sites and regulate gene transcription.

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Caffeine inhibits gene conversion by displacing Rad51 from ssDNA.

Project description:Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1(ATR)/Tel1(ATM)-dependent DNA damage response or caffeine's inhibition of 5' to 3' resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.

| S-EPMC4538809 | biostudies-literature

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