Avoiding chromosome pathology when replication forks collide

Rudolph, Christian J.; Upton, Amy L.; Stockum, Anna; Nieduszynski, Conrad A.; Lloyd, Robert G.

doi:10.1038/nature12312

Cited by 120 publications

(445 citation statements)

References 30 publications

(47 reference statements)

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“…3C) (38). In both recD and recG mutants, the DNA ends from unresolved completion events lead to over-replication that can also be observed on plasmids.…”

Section: Resultsmentioning

confidence: 99%

Completion of DNA replication in Escherichia coli

Wendel

Courcelle

2014

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

134

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The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.replication completion | double-strand break repair | RecBCD | homologous recombination | SbcDC

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“…3C) (38). In both recD and recG mutants, the DNA ends from unresolved completion events lead to over-replication that can also be observed on plasmids.…”

Section: Resultsmentioning

confidence: 99%

Completion of DNA replication in Escherichia coli

Wendel

Courcelle

2014

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

134

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“…The ATPase activity of RadD was necessary for this effect, with an ATPase-dead RadD displaying a dominant negative effect in response to UV. This role also showed overlap with the functions of RadA, a RecA paralogue involved in homologous recombination (22,23), and RecG, a superfamily 2 helicase involved in stabilizing recombination intermediates and preventing origin-independent replication (24,25).…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

Chen

Byrne-Nash

Cox

2016

Journal of Biological Chemistry

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The bacterial single-stranded DNA binding protein (SSB) acts as an organizer of DNA repair complexes. The radD gene was recently identified as having an unspecified role in repair of radiation damage and, more specifically, DNA double-strand breaks. Purified RadD protein displays a DNA-independent ATPase activity. However, ATP hydrolytic rates are stimulated by SSB through its C terminus. The RadD and SSB proteins also directly interact in vivo in a yeast two-hybrid assay and in vitro through ammonium sulfate co-precipitation. Therefore, it is likely that the repair function of RadD is mediated through interaction with SSB at the site of damage.In Escherichia coli the single-stranded DNA-binding protein (SSB) 2 functions as a molecular organizer of a variety of DNA replication and repair functions (1). Nearly a score of different proteins directly interact with the C-terminal eight amino acids of SSB (1-9), and newly discovered members of the SSB interactome are being described regularly. In some cases the activity of the interacting protein is stimulated by SSB. All of the proteins that interact with SSB to date have a role in some aspect of DNA metabolism. Therefore, an interaction with the C terminus of SSB provides an increasingly reliable indicator of DNA metabolic function.The radD gene (formerly yejH) was recently implicated in the repair of DNA double-strand breaks after radiation or chemical damage (10,11). The peptide sequence of the RadD protein includes all seven of the motifs associated with a superfamily 2 (SF2) helicase (10). Although there is little homology outside of these regions, the helicase motifs align well to other E. coli SF2 helicases including RecG and RecQ. RadD also contains a putative zinc finger motif in the C terminus that could aid in binding to DNA substrates. Both the ATPase and zinc binding domains are necessary for the rescue of DNA repair activities of radD deletion strains in vivo (10).Here, we present the first biochemical characterization of the RadD protein. Although helicase activity was not observed, RadD hydrolyzes ATP in the absence of DNA. This activity is stimulated by the SSB protein, specifically the C-terminal amino acid residues of SSB. The RadD and SSB proteins interact in vivo and directly in vitro. The previous study and this work, taken together, suggest that SSB likely recruits and activates RadD in the cell during DNA repair. ResultsRationale-Based on the sequence similarity of RadD to known DNA metabolism proteins and a recent study implicating RadD in the repair of double-strand DNA breaks (10), we natively purified the RadD protein and screened for likely activities and interactions. A number of assays for helicase activity, including a variety of both DNA and RNA substrates, produced no positive results and thus are not reported. Presented here are results that demonstrate DNA-independent ATPase activity for RadD as well as an interaction with the SSB C terminus.RadD Protein Has a DNA-independent ATPase Activity-As a predicted SF2 helicase (10), Rad...

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“…However, oriM2 has not been mapped accurately and it is not known whether the DSB that we observe relates to this origin. Several other results, such as the existence of terminal recombination (30)(31)(32) and the striking replication profile of a recB mutant (33), indicate that the terminus region of the chromosome presents an area of importance to recombination. However, there are very likely several different reactions taking place.…”

Section: Discussionmentioning

confidence: 99%

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Cockram

Milana

Danos

et al. 2015

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

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Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/ nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.homologous recombination | mechanistic modelling | DNA repair | RecA | RecBCD

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Avoiding chromosome pathology when replication forks collide

Cited by 120 publications

References 30 publications

Completion of DNA replication in Escherichia coli

Completion of DNA replication in Escherichia coli

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

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