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Journal ArticleDOI

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins.

TL;DR: In this article, the authors employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RFS-1/RIP-1.
About: This article is published in Molecular Cell.The article was published on 2021-03-04 and is currently open access. It has received 40 citations till now. The article focuses on the topics: Strand invasion & Replication protein A.
Citations
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Journal ArticleDOI
TL;DR: In this article, the authors used single-molecule imaging to reveal that the Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone.

37 citations

Journal ArticleDOI
TL;DR: In this article , it was shown that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

12 citations

Journal ArticleDOI
TL;DR: In this article , the authors review recent advances in the ability to characterise biomolecular structure, interactions and associated dynamics by mass photometry (MP), the label-free detection and mass measurement of individual biomolecules in solution.

8 citations

Journal ArticleDOI
TL;DR: Several mechanistic commonalities for DNA damage detection were discovered with these techniques, including a combination of 3-dimensional and linear diffusion for surveying damaged sites within long stretches of DNA as mentioned in this paper.
Abstract: DNA is under constant threat of damage from a variety of chemical and physical insults, such as ultraviolet rays produced by sunlight and reactive oxygen species produced during respiration or inflammation. Because damaged DNA, if not repaired, can lead to mutations or cell death, multiple DNA repair pathways have evolved to maintain genome stability. Two repair pathways, nucleotide excision repair (NER) and base excision repair (BER), must sift through large segments of nondamaged nucleotides to detect and remove rare base modifications. Many BER and NER proteins share a common base-flipping mechanism for the detection of modified bases. However, the exact mechanisms by which these repair proteins detect their damaged substrates in the context of cellular chromatin remains unclear. The latest generation of single-molecule techniques, including the DNA tightrope assay, atomic force microscopy, and real-time imaging in cells, now allows for nearly direct visualization of the damage search and detection processes. This review describes several mechanistic commonalities for damage detection that were discovered with these techniques, including a combination of 3-dimensional and linear diffusion for surveying damaged sites within long stretches of DNA. We also discuss important findings that DNA repair proteins within and between pathways cooperate to detect damage. Finally, future technical developments and single-molecule studies are described which will contribute to the growing mechanistic understanding of DNA damage detection.

7 citations

Journal ArticleDOI
TL;DR: It is built upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions, and provides a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.
Abstract: Abstract SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B–RAD51C–RAD51D–XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.

7 citations

References
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Journal ArticleDOI
24 Oct 2003-Science
TL;DR: Physical exercise and lack of obesity in adolescence were associated with significantly delayed breast cancer onset, and risks appear to be increasing with time.
Abstract: Risks of breast and ovarian cancer were determined for Ashkenazi Jewish women with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2. We selected 1008 index cases, regardless of family history of cancer, and carried out molecular analysis across entire families. The lifetime risk of breast cancer among female mutation carriers was 82%, similar to risks in families with many cases. Risks appear to be increasing with time: Breast cancer risk by age 50 among mutation carriers born before 1940 was 24%, but among those born after 1940 it was 67%. Lifetime risks of ovarian cancer were 54% for BRCA1 and 23% for BRCA2 mutation carriers. Physical exercise and lack of obesity in adolescence were associated with significantly delayed breast cancer onset.

2,043 citations

Journal ArticleDOI
06 Mar 2014-Nature
TL;DR: It is shown that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM) and that PAM interactions trigger Cas9 catalytic activity.
Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

1,621 citations

Journal ArticleDOI
TL;DR: Recent insights are reviewed into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.

1,427 citations

Journal ArticleDOI
13 May 2011-Cell
TL;DR: Using single-molecule DNA fiber analysis, it is shown that nascent replication tracts created before fork stalling with hydroxyurea are degraded in the absence of BRCA2 but are stable in wild-type cells.

1,001 citations

Journal ArticleDOI
17 Sep 2004-Cell
TL;DR: 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.

938 citations