RecA finds homologous DNA by reduced dimensionality search

AbstractHomologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

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DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

Nature Communications ◽

10.1038/s41467-021-24341-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Prasun Chakraborty ◽

Kevin Hiom

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Rna Polymerase Ii ◽

Critical Role ◽

Dna Breaks ◽

Double Stranded Dna ◽

Recombination Repair ◽

Dna End Resection ◽

End Resection ◽

Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

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Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

10.1101/704197 ◽

2019 ◽

Author(s):

Geoffrey L. Rogers ◽

Hsu-Yu Chen ◽

Heidy Morales ◽

Paula M. Cannon

Keyword(s):

Homologous Recombination ◽

Progenitor Cells ◽

Genome Editing ◽

High Efficiency ◽

Zinc Finger Nucleases ◽

Dna Breaks ◽

Adeno Associated Virus ◽

Hematopoietic Stem ◽

Double Stranded Dna ◽

Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

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Direct, sensitive and specific detection of individual single- or double-strand DNA breaks by fluorescence microscopy

10.1101/772269 ◽

2019 ◽

Author(s):

Magdalena Kordon ◽

Mirosław Zarębski ◽

Kamil Solarczyk ◽

Hanhui Ma ◽

Thoru Pederson ◽

...

Keyword(s):

Dna Damage ◽

Fluorescence Microscopy ◽

Single Cells ◽

Primary Cultures ◽

Dna Lesions ◽

Dna Breaks ◽

Damage Site ◽

Double Strand Dna Breaks ◽

Age Related ◽

Double Strand Dna

ABSTRACTWe here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. Sensitivity of STRIDE was tested using specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used TUNEL assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions), and fragmentation of DNA in human spermatozoa. STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

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RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity

10.1101/2021.10.04.463068 ◽

2021 ◽

Author(s):

Alex L. Payne-Dwyer ◽

Aisha H. Syeda ◽

Jack W. Shepherd ◽

Lewis Frame ◽

Mark. C. Leake

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Dna Repair ◽

Mitomycin C ◽

Single Molecule ◽

Reca Protein ◽

Live Cells ◽

Dna Breaks ◽

Fluorescent Reporters ◽

Double Stranded Dna

AbstractThe RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA, RecBCD a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks and also facilitating decoration of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA and the RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting changes in stoichiometry, copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA filamental stoichiometries equivalent to several hundred molecules that act largely in RecA tetramers before DNA damage, but switch to approximately hexameric subunits when mature filaments are formed. Unexpectedly, we find that the physiologically predominant form of RecB is a dimer.

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RAD51 Is Required for Propagation of the Germinal Nucleus in Tetrahymena thermophila

Genetics ◽

10.1093/genetics/154.4.1587 ◽

2000 ◽

Vol 154 (4) ◽

pp. 1587-1596 ◽

Cited By ~ 1

Author(s):

Thomas C Marsh ◽

Eric S Cole ◽

Kathleen R Stuart ◽

Colin Campbell ◽

Daniel P Romero

Keyword(s):

Escherichia Coli ◽

Cell Cycle ◽

Dna Damage ◽

Gene Disruption ◽

Tetrahymena Thermophila ◽

S Phase ◽

Dna Breaks ◽

Double Stranded Dna ◽

Meiosis I

Abstract RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I). We discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.

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sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00613-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7562-7571 ◽

Cited By ~ 8

Author(s):

Ksenija Zahradka ◽

Sanela Šimić ◽

Maja Buljubašić ◽

Mirjana Petranović ◽

Damir Đermić ◽

...

Keyword(s):

Escherichia Coli ◽

Recq Helicase ◽

Dna Breaks ◽

Γ Irradiation ◽

E Coli ◽

Exonuclease I ◽

Double Stranded Dna ◽

Recombination Processes ◽

Recf Pathway ◽

Recombination Reactions

ABSTRACT Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.

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Single molecule approaches to monitor the recognition and resection of double-stranded DNA breaks during homologous recombination

DNA Repair ◽

10.1016/j.dnarep.2014.02.002 ◽

2014 ◽

Vol 20 ◽

pp. 119-129 ◽

Cited By ~ 10

Author(s):

Carolina Carrasco ◽

Mark S. Dillingham ◽

Fernando Moreno-Herrero

Keyword(s):

Homologous Recombination ◽

Single Molecule ◽

Dna Breaks ◽

Double Stranded Dna

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Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase

Communications Biology ◽

10.1038/s42003-021-02690-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Prajwal C. Boddu ◽

Abhishek K. Gupta ◽

Jung-Sik Kim ◽

Karla M. Neugebauer ◽

Todd Waldman ◽

...

Keyword(s):

Homologous Recombination ◽

Mutant Allele ◽

Cre Recombinase ◽

Splicing Factor ◽

Splicing Factors ◽

Dna Breaks ◽

Genomic Locus ◽

Trap Design ◽

Double Stranded Dna ◽

Cancer Models

AbstractScalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.

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Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4493-4500.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4493-4500

Author(s):

R H Schiestl ◽

J Zhu ◽

T D Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Restriction Enzyme ◽

Strand Break ◽

Illegitimate Recombination ◽

Dna Breaks ◽

Homologous Integration ◽

Double Stranded Dna ◽

Fold Reduction

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.

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