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Open accessJournal ArticleDOI: 10.1016/J.MOLCEL.2020.12.019

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination.

04 Mar 2021-Molecular Cell (Cell Press)-Vol. 81, Iss: 5
Abstract: Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.

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Topics: RAD51 (57%), Homologous recombination (52%), DNA repair (51%) ... read more
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16 results found


Open accessPosted ContentDOI: 10.1101/2021.09.30.462231
Ian R. Humphreys1, Jimin Pei2, Minkyung Baek1, Krishnakumar A1  +26 moreInstitutions (12)
30 Sep 2021-bioRxiv
Abstract: Protein-protein interactions play critical roles in biology, but despite decades of effort, the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions that have not yet been identified. Here, we take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes, as represented within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of S. cerevisiae proteins and build models for strongly predicted protein assemblies with two to five components. Comparison to existing interaction and structural data suggests that these predictions are likely to be quite accurate. We provide structure models spanning almost all key processes in Eukaryotic cells for 104 protein assemblies which have not been previously identified, and 608 which have not been structurally characterized. One-sentence summary We take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes.

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7 Citations


Open accessJournal ArticleDOI: 10.1126/SCIENCE.ABM4805
Ian R. Humphreys1, Jimin Pei2, Minkyung Baek1, Aditya Krishnakumar1  +28 moreInstitutions (12)
11 Nov 2021-Science
Abstract: Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take ...

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2 Citations


Journal ArticleDOI: 10.1016/J.GDE.2021.06.010
Abstract: RAD51 paralog gene mutations are observed in both hereditary breast and ovarian cancers. Classically, defects in RAD51 paralog function are associated with homologous recombination (HR) deficiency and increased genomic instability. Several recent investigative advances have enabled characterization of non-canonical RAD51 paralog function during DNA replication. Here we discuss the role of the RAD51 paralogs and their associated complexes in integrating a robust response to DNA replication stress. We highlight recent discoveries suggesting that the RAD51 paralogs complexes mediate lesion-specific tolerance of replicative stress following exposure to alkylating agents and the requirement for the Shu complex in fork restart upon fork stalling by dNTP depletion. In addition, we describe the role of the BCDX2 complex in restraining and promoting fork remodeling in response to fluctuating dNTP pools. Finally, we highlight recent work demonstrating a requirement for RAD51C in recognizing and tolerating methyl-adducts. In each scenario, RAD51 paralog complexes play a central role in lesion recognition and bypass in a replicative context. Future studies will determine how these critical functions for RAD51 paralog complexes contribute to tumorigenesis.

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Topics: RAD51 (52%), Gene mutation (51%), DNA replication (51%) ... read more

2 Citations


Journal ArticleDOI: 10.1016/J.MOLCEL.2021.01.037
Petr Cejka1Institutions (1)
04 Mar 2021-Molecular Cell
Abstract: In this issue of Molecular Cell, Roy et al. (2021) and Belan et al. (2021) demonstrate that the yeast and nematode RAD51 paralog complexes function as chaperones to promote the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA.

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2 Citations


Journal ArticleDOI: 10.1016/J.DNAREP.2021.103114
07 Jun 2021-DNA Repair
Abstract: DNA is the molecule that stores the chemical instructions necessary for life and its stability is therefore of the utmost importance. Despite this, DNA is damaged by both exogenous and endogenous factors at an alarming frequency. The most severe type of DNA damage is a double-strand break (DSB), in which a scission occurs in both strands of the double helix, effectively dividing a single normal chromosome into two pathological chromosomes. Homologous recombination (HR) is a universal DSB repair mechanism that solves this problem by identifying another region of the genome that shares high sequence similarity with the DSB site and using it as a template for repair. Rad51 possess the enzymatic activity that is essential for this repair but several auxiliary factors are required for Rad51 to fulfil its function. It is becoming increasingly clear that many HR factors are subjected to post-translational modification. Here, we review what is known about how these modifications affect HR. We first focus on cases where there is experimental evidence to support a function for the modification, then discuss speculative cases where a function can be inferred. Finally, we contemplate why such modifications might be necessary.

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Topics: DNA repair (56%), RAD51 (55%), Homologous recombination (53%) ... read more

1 Citations


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58 results found


Book ChapterDOI: 10.1016/S0076-6879(02)50954-X
Fred Sherman1Institutions (1)
Abstract: Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

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Topics: Yeast artificial chromosome (57%), Saccharomyces cerevisiae (57%), Yeast (57%) ... read more

3,476 Citations


Journal ArticleDOI: 10.1016/0092-8674(89)90584-9
Barbara J. Thomas1, Rodney Rothstein1Institutions (1)
24 Feb 1989-Cell
Abstract: We have examined the effect of RNA polymerase II-dependent transcription on recombination between directly repeated sequences of the GAL10 gene in S. cerevisiae. Direct repeat recombination leading either to plasmid loss or conversion was examined in isogenic strains containing null mutations in the positive activator, GAL4, or the repressor, GAL80. A 15-fold increase in the rate of plasmid loss is observed in cells constitutively expressing the construct compared with cells that are not. Conversion events that retain the integrated plasmid are not stimulated by expression of the repeats. Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmid sequences.

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Topics: Plasmid (58%), Direct repeat (55%), Repressor (55%) ... read more

1,602 Citations


Abstract: Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of repli- cation forks, for telomere maintenance, and chromosome segrega- tion in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facil- itate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.

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Topics: RAD51 (63%), Non-homologous end joining (60%), Homologous recombination (59%) ... read more

1,406 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELL.2004.08.015
17 Sep 2004-Cell
Abstract: DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

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Topics: DNA repair (66%), G2-M DNA damage checkpoint (66%), Replication protein A (65%) ... read more

908 Citations


Journal ArticleDOI: 10.1038/NG.569
Alfons Meindl1, Heide Hellebrand1, Constanze Wiek2, Verena Erven2  +21 moreInstitutions (9)
01 May 2010-Nature Genetics
Abstract: Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia-like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.

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Topics: Germline mutation (60%), Cancer (58%), Breast cancer (58%) ... read more

647 Citations


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202116