Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks

1 AbstractThe bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing double-stranded DNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54-kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella spp. and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the E. coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

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Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones

eLife ◽

10.7554/elife.22963 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 20

Author(s):

Martin Wilkinson ◽

Lucy Troman ◽

Wan AK Wan Nur Ismah ◽

Yuriy Chaban ◽

Matthew B Avison ◽

...

Keyword(s):

Molecular Mimicry ◽

Synthetic Lethality ◽

Fluoroquinolone Resistance ◽

Structural Basis ◽

Dna Breaks ◽

E Coli ◽

Double Stranded Dna ◽

Dna Binding Site ◽

Interaction Surface ◽

Dna Substrate

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.

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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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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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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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Crystal Structure ofDeinococcus radioduransRecQ Helicase Catalytic Core Domain: The Interdomain Flexibility

BioMed Research International ◽

10.1155/2014/342725 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Sheng-Chia Chen ◽

Chi-Hung Huang ◽

Chia Shin Yang ◽

Tzong-Der Way ◽

Ming-Chung Chang ◽

...

Keyword(s):

Crystal Structure ◽

Deinococcus Radiodurans ◽

Domain Architecture ◽

Genome Integrity ◽

Catalytic Core Domain ◽

Catalytic Core ◽

Core Domain ◽

Winged Helix ◽

Dna Helicases ◽

E Coli

RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ fromDeinococcus radiodurans(DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of theE. coliRecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.

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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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