Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis.

ABSTRACT: Meiotic recombination in budding yeast requires two RecA-related proteins, Rad51 and Dmc1, both of which form filaments on DNA capable of directing homology search and catalyzing formation of homologous joint molecules (JMs) and strand exchange. With use of a separation-of-function mutant form of Rad51 that retains filament-forming but not JM-forming activity, we show that the JM activity of Rad51 is fully dispensable for meiotic recombination. The corresponding mutation in Dmc1 causes a profound recombination defect, demonstrating Dmc1's JM activity alone is responsible for meiotic recombination. We further provide biochemical evidence that Rad51 acts with Mei5-Sae3 as a Dmc1 accessory factor. Thus, Rad51 is a multifunctional protein that catalyzes recombination directly in mitosis and indirectly, via Dmc1, during meiosis.

SUBMITTER: Cloud V

PROVIDER: S-EPMC4056682 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis.

Cloud Veronica V Chan Yuen-Ling YL Grubb Jennifer J Budke Brian B Bishop Douglas K DK

Science (New York, N.Y.) 20120901 6099

Meiotic recombination in budding yeast requires two RecA-related proteins, Rad51 and Dmc1, both of which form filaments on DNA capable of directing homology search and catalyzing formation of homologous joint molecules (JMs) and strand exchange. With use of a separation-of-function mutant form of Rad51 that retains filament-forming but not JM-forming activity, we show that the JM activity of Rad51 is fully dispensable for meiotic recombination. The corresponding mutation in Dmc1 causes a profoun ...[more]

PMID: 22955832

Similar Datasets

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase.

Project description:Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.

| S-EPMC7260962 | biostudies-literature

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation.

Project description:Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.

| S-EPMC3711438 | biostudies-literature

DMC1 attenuates RAD51-mediated recombination in Arabidopsis.

Project description:Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.

| S-EPMC9451096 | biostudies-literature

Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks.

Project description:Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.

| S-EPMC3900393 | biostudies-literature

RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination.

Project description:Dmc1 catalyzes homology search and strand exchange during meiotic recombination in budding yeast and many other organisms including humans. Here we reconstitute Dmc1 recombination in vitro using six purified proteins from budding yeast including Dmc1 and its accessory proteins RPA, Rad51, Rdh54/Tid1, Mei5-Sae3 and Hop2-Mnd1 to promote D-loop formation between ssDNA and dsDNA substrates. Each accessory protein contributed to Dmc1's activity, with the combination of all six proteins yielding optimal activity. The ssDNA binding protein RPA plays multiple roles in stimulating Dmc1's activity including by overcoming inhibitory effects of ssDNA secondary structure on D-loop reactions, and by elongating D-loops. In addition, we demonstrate that RPA limits inhibitory interactions of Hop2-Mnd1 and Rdh54/Tid1 that otherwise occur during assembly of Dmc1-ssDNA nucleoprotein filaments. Finally, we report interactions between the proteins employed in the biochemical reconstitution including a direct interaction between Rad51 and Dmc1 that is enhanced by Mei5-Sae3.

| S-EPMC6344864 | biostudies-other

The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.

Project description:Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

| S-EPMC7447979 | biostudies-literature

Dmc1 is a candidate for temperature tolerance during wheat meiosis.

Project description:Key messageThe meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures. We initially assessed the effects of low temperature on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (Triticum aestivum L.) variety 'Chinese Spring'. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene (Ltp1) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low temperature in a controlled environment room during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after 7 days at 13 °C, suggesting that Ltp1 is deleted in this mutant. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ltp1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. KASP genotyping revealed that the ltp1 mutant has a 4-Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low temperatures is the meiotic recombination gene Dmc1. The ltp1 mutants were subsequently treated at 30 °C for 24 h during meiosis and exhibited a reduced number of crossovers and increased univalence, though to a lesser extent than at 13 °C. We therefore renamed our ltp1 mutant 'ttmei1' (temperature-tolerant meiosis 1) to reflect this additional loss of high temperature tolerance.

| S-EPMC7021665 | biostudies-literature

A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability.

Project description:During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes ("homologs"), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1's activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.

| S-EPMC6907854 | biostudies-literature

Hop2 and Sae3 Are Required for Dmc1-Mediated Double-Strand Break Repair via Homolog Bias during Meiosis.

Project description:During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the hop2Δ or sae3Δ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation.

| S-EPMC4959020 | biostudies-literature

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.

Project description:During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

| S-EPMC4267644 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data