Interphase Chromatin Dynamics in Response to Double Stranded DNA Breaks

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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A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽

10.1093/genetics/153.2.595 ◽

1999 ◽

Vol 153 (2) ◽

pp. 595-605 ◽

Cited By ~ 2

Author(s):

Bradley J Merrill ◽

Connie Holm

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Synthetic Lethality ◽

Velocity Sedimentation ◽

Recombinational Repair ◽

Repair Pathway ◽

Dna Breaks ◽

Single Stranded Dna ◽

Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast

10.1101/131995 ◽

2017 ◽

Author(s):

Yi Yin ◽

Margaret Dominska ◽

Eunice Yim ◽

Thomas D. Petes

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Mitotic Recombination ◽

Template Switching ◽

Wild Type ◽

Dna Breaks ◽

Repair Synthesis ◽

Heteroduplex Dna ◽

Dna Molecule ◽

Double Stranded Dna

AbstractDouble-stranded DNA breaks (DSBs) can be generated by both endogenous and exogenous agents. In diploid yeast strains, such breaks are usually repaired by homologous recombination (HR), and a number of different HR pathways have been described. An early step for all HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, within the heteroduplex DNA (hetDNA), there will be mismatches. In a wild-type strain, these mismatches are removed by the mismatch repair (MMR) system. In strains lacking MMR, the mismatches persist and can be detected by a variety of genetic and physical techniques. Most previous studies involving hetDNA formed during mitotic recombination have been restricted to a single locus with DSBs induced at a defined position by a site-specific endonuclease. In addition, in most of these studies, recombination between repeated genes was examined; in such studies, the sequence homologies were usually less than 5 kb. In the present study, we present a global mapping of hetDNA formed in a UV-treated MMR-defective mlh1 strain. Although about two-thirds of the recombination events were associated with hetDNA with a continuous array of unrepaired mismatches, in about one-third of the events, we found regions of unrepaired mismatches flanking regions without mismatches. We suggest that these discontinuous hetDNAs involve template switching during repair synthesis, repair of a double-stranded DNA gap, and/or Mlh1-independent MMR. Many of our observed events are not explicable by the simplest form of the double-strand break repair (DSBR) model of recombination. We also studied hetDNA associated with spontaneous recombination events selected on chromosomes IV and V in a wild-type strain. The interval on chromosome IV contained a hotspot for spontaneous crossovers generated by an inverted pair of transposable elements (HS4). We showed that HS4-induced recombination events are associated with the formation of very large (>30 kb) double-stranded DNA gaps.

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CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

10.1101/2021.03.10.434760 ◽

2021 ◽

Author(s):

Long Jiang ◽

Katrine Ingelshed ◽

Yunbing Shen ◽

Sanjaykumar V. Boddul ◽

Vaishnavi Srinivasan Iyer ◽

...

Keyword(s):

Cellular Response ◽

Human Cancer ◽

Genetic Alterations ◽

Dna Breaks ◽

Data Set ◽

Human Cancer Cell Lines ◽

Cycle Arrest ◽

Double Stranded Dna ◽

A Cell

CRISPR/Cas9 can be used to inactivate or modify genes by inducing double-stranded DNA breaks1–3. As a protective cellular response, DNA breaks result in p53-mediated cell cycle arrest and activation of cell death programs4,5. Inactivating p53 mutations are the most commonly found genetic alterations in cancer, highlighting the important role of the gene6–8. Here, we show that cells deficient in p53, as well as in genes of a core CRISPR-p53 tumor suppressor interactome, are enriched in a cell population when CRISPR is applied. Such enrichment could pose a challenge for clinical CRISPR use. Importantly, we identify that transient p53 inhibition suppresses the enrichment of cells with these mutations. Furthermore, in a data set of >800 human cancer cell lines, we identify parameters influencing the enrichment of p53 mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, our data identify strategies enabling safe CRISPR use.

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Mechanism for nuclease regulation in RecBCD

eLife ◽

10.7554/elife.18227 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 16

Author(s):

Martin Wilkinson ◽

Yuriy Chaban ◽

Dale B Wigley

Keyword(s):

Nuclease Activity ◽

Binding Mode ◽

Sh3 Domain ◽

Bacterial Cells ◽

Dna Breaks ◽

Nuclease Domain ◽

Double Stranded Dna ◽

Large Conformational Change ◽

Enzyme Complexes ◽

Dna Substrate

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5’-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.

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Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽

10.1128/mbio.00342-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhiwei Hu ◽

Yannan Wang ◽

Qian Liu ◽

Yan Qiu ◽

Zhiyu Zhong ◽

...

Keyword(s):

Genome Editing ◽

Dna Breaks ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rna ◽

Guide Rnas ◽

Double Stranded Dna ◽

Target Sites ◽

Guide Sequence ◽

Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

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Quantification of Double Stranded DNA Breaks and Telomere Length as Proxies for Corneal Damage and Replicative Stress in Human Keratoconus Corneas

Translational Vision Science & Technology ◽

10.1167/tvst.8.4.10 ◽

2019 ◽

Vol 8 (4) ◽

pp. 10

Author(s):

Robert P. L. Wisse ◽

Jonas J. W. Kuiper ◽

Timothy R. D. Radstake ◽

Jasper C. A. Broen

Keyword(s):

Telomere Length ◽

Dna Breaks ◽

Replicative Stress ◽

Double Stranded Dna ◽

Corneal Damage

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The Human T-Cell Leukemia Virus Type 1 Basic Leucine Zipper Factor Attenuates Repair of Double-Stranded DNA Breaks via Nonhomologous End Joining

Journal of Virology ◽

10.1128/jvi.00672-18 ◽

2018 ◽

Vol 92 (15) ◽

Cited By ~ 8

Author(s):

Amanda W. Rushing ◽

Kimson Hoang ◽

Nicholas Polakowski ◽

Isabelle Lemasson

Keyword(s):

T Cell ◽

Leukemia Virus ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Dna Breaks ◽

Dsb Repair ◽

Cell Leukemia Virus Type ◽

Double Stranded Dna ◽

Human T Cell ◽

T Cell Leukemia Virus

ABSTRACTAdult T-cell leukemia (ATL) is a fatal malignancy of CD4+T cells infected with human T-cell leukemia virus type 1 (HTLV-1). ATL cells often exhibit random gross chromosomal rearrangements that are associated with the induction and improper repair of double-stranded DNA breaks (DSBs). The viral oncoprotein Tax has been reported to impair DSB repair but has not been shown to be consistently expressed throughout all phases of infection. The viral oncoprotein HTLV-1 basic leucine zipper (bZIP) factor (HBZ) is consistently expressed prior to and throughout disease progression, but it is unclear whether it also influences DSB repair. We report that HBZ attenuates DSB repair by nonhomologous end joining (NHEJ), in a manner dependent upon the bZIP domain. HBZ was found to interact with two vital members of the NHEJ core machinery, Ku70 and Ku80, and to be recruited to DSBs in a bZIP-dependent mannerin vitro. We observed that HBZ expression also resulted in a bZIP-dependent delay in DNA protein kinase (DNA-PK) activation following treatment with etoposide. Although Tax is reported to interact with Ku70, we did not find Tax expression to interfere with HBZ:Ku complex formation. However, as Tax was reported to saturate NHEJ, we found that this effect masked the attenuation of NHEJ by HBZ. Overall, these data suggest that DSB repair mechanisms are impaired not only by Tax but also by HBZ and show that HBZ expression may significantly contribute to the accumulation of chromosomal abnormalities during HTLV-1-mediated oncogenesis.IMPORTANCEHuman T-cell leukemia virus type 1 (HTLV-1) infects 15 million to 20 million people worldwide. Approximately 90% of infected individuals are asymptomatic and may remain undiagnosed, increasing the risk that they will unknowingly transmit the virus. About 5% of the HTLV-1-positive population develop adult T-cell leukemia (ATL), a fatal disease that is not highly responsive to treatment. Although ATL development remains poorly understood, two viral proteins, Tax and HBZ, have been implicated in driving disease progression by manipulating host cell signaling and transcriptional pathways. Unlike Tax, HBZ expression is consistently observed in all infected individuals, making it important to elucidate the specific role of HBZ in disease progression. Here, we present evidence that HBZ could promote the accumulation of double-stranded DNA breaks (DSBs) through the attenuation of the nonhomologous end joining (NHEJ) repair pathway. This effect may lead to genome instability, ultimately contributing to the development of ATL.

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Nonhomologous end-joining repair is likely involved in the repair of double-stranded DNA breaks induced by riluzole in melanoma cells

Melanoma Research ◽

10.1097/cmr.0000000000000652 ◽

2020 ◽

Vol 30 (3) ◽

pp. 303-308

Author(s):

Robert Cerchio ◽

Christina Marinaro ◽

Tzeh Keong Foo ◽

Bing Xia ◽

Suzie Chen

Keyword(s):

Melanoma Cells ◽

Nonhomologous End Joining ◽

Dna Breaks ◽

End Joining ◽

Double Stranded Dna

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Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518028112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6852-E6861 ◽

Cited By ~ 23

Author(s):

Behzad Rad ◽

Anthony L. Forget ◽

Ronald J. Baskin ◽

Stephen C. Kowalczykowski

Keyword(s):

Single Molecule ◽

Fluorescent Sensor ◽

Holliday Junctions ◽

Dna Unwinding ◽

Recq Helicase ◽

Dna Breaks ◽

Dna Helicases ◽

Double Stranded Dna ◽

Functional Forms ◽

And Migration

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

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