RecA bundles mediate homology pairing between distant sisters during DNA break repair

Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

doi:10.1038/nature12868

Cited by 162 publications

(290 citation statements)

References 43 publications

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“…The peripheral localization of transcription is reminiscent of that of DNA repair (45), supporting the view that DNA metabolism activities involving large machineries locate preferentially at the nucleoid periphery, where large protein complex formation is facilitated. In rich media conditions, the number of RNAP clusters observed per cell (10), and the number of RNAPs found in each cluster (4), has led to the hypothesis that these clusters represent RNAPs active on multiple heavily transcribed genes spatially located to the same site, analogous to transcription factories in eukaryotes (11,46).…”

Section: Discussionsupporting

confidence: 51%

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

Stracy

Lesterlin

Leon

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.RNA polymerase | transcription | superresolution | single-molecule tracking | protein-DNA interactions

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Section: Discussionsupporting

confidence: 51%

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

Stracy

Lesterlin

Leon

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

241

346

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“…Previous work in E. coli and B. subtilis examined the ability of RecA-GFP to organize into foci (5)(6)(7)(8)(9)(10)(11)(12)43). Prior experiments have tested RecA-GFP as the only source of RecA in vivo (6,8,9,38) GFP-RecA was expressed ectopically as the sole source of RecA in cells or in merodiploid cells with the native recA allele intact (5,12).…”

Section: Resultsmentioning

confidence: 99%

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

et al. 2014

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bRecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify singlestranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.

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“…5A). We assume that these filaments are the same as the RecA-GFP "threads" observed by others after UV exposure and during DSB repair (71)(72)(73). These threads have been hypothesized to be either the RecA/single-stranded DNA (ssDNA) nucleoprotein filaments that initiate recombination and/or RecA bound to ssDNA gaps left when replication reinitiates downstream of a DNA lesion (71)(72)(73).…”

Section: Discussionmentioning

confidence: 99%

“…8A). Since RecA processes DSBs, we assume, as have others (48,72), that the RecA-GFP foci are actually at the DSBs. During normal cell growth, DSBs are common and, if unrepaired, are lethal.…”

Section: Discussionmentioning

confidence: 99%

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

et al. 2015

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Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNAdamaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ϳ60% of the foci consisted of three Pol IV molecules, while ϳ40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ϳ70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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RecA bundles mediate homology pairing between distant sisters during DNA break repair

Cited by 162 publications

References 43 publications

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

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