RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms

Courcelle, Justin; Wendel, Brian M.; Livingstone, Dena D.; Courcelle, Charmain T.

doi:10.1016/j.dnarep.2015.04.018

Cited by 38 publications

(78 citation statements)

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“…The amplification of terminal DNA in a recG mutant has been examined in detail [6, 34, 39, 40], while the DNA loss in the recB mutant has been less extensively explored, and the model of merging forks has not been directly tested experimentally [35, 41]. Here we use Marker Frequency Analysis (MFA) and live-cell fluorescence microscopy to further characterize this phenomenon.…”

Section: Introductionmentioning

confidence: 99%

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

et al. 2017

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Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.

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Section: Introductionmentioning

confidence: 99%

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

et al. 2017

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“…Failure of even a single completion event would be expected to result in duplications, deletions, rearrangements, or cell death, depending on how the convergent strands are processed. Cells devote a large number of enzymes to maintain the fidelity of both replication initiation and elongation, and recent studies have shown that this final step, required to maintain genomic integrity, similarly requires enzymatic control and is tightly regulated (3,4).…”

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confidence: 99%

“…In E. coli, the completion of DNA replication requires the RecBCD helicase-nuclease complex and occurs through a reaction that does not involve recombination or RecA (3,4). In the absence of RecBCD, cultures either fail to replicate or fail to maintain the chromosome region where replication forks converge, leading to large segments of the chromosome remaining unreplicated or degraded in this region.…”

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confidence: 99%

SbcC-SbcD and ExoI process convergent forks to complete chromosome replication

Wendel

Cole

Courcelle

et al. 2017

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

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SbcC-SbcD are the bacterial orthologs of Mre11-Rad50, a nuclease complex essential for genome stability, normal development, and viability in mammals. In vitro, these enzymes degrade long DNA palindromic structures. When inactivated along with ExoI in , or Sae2 in eukaryotes, palindromic amplifications arise and propagate in cells. However, long DNA palindromes are not normally found in bacterial or human genomes, leaving the cellular substrates and function of these enzymes unknown. Here, we show that during the completion of DNA replication, convergent replication forks form a palindrome-like structural intermediate that requires nucleolytic processing by SbcC-SbcD and ExoI before chromosome replication can be completed. Inactivation of these nucleases prevents completion from occurring, and under these conditions, cells maintain viability by shunting the reaction through an aberrant recombinational pathway that leads to amplifications and instability in this region. The results identify replication completion as an event critical to maintain genome integrity and cell viability, demonstrate SbcC-SbcD-ExoI acts before RecBCD and is required to initiate the completion reaction, and reveal how defects in completion result in genomic instability.

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“…RecBCD is one of the most fascinating and studied multienzymatic complexes in bacteria. It is the major recombinational pathway in E. coli responsible for the repair of DSB, conjugational recombination, but also for the degradation of foreign linear DNA, and recently RecBCD was shown to participate in completion of DNA replication (41,42). We unravel here that, in addition to these many functions, RecBCD also plays a role in SSG repair.…”

Section: Discussionmentioning

confidence: 80%

A non-catalytic role of RecBCD in Homology Directed Gap Repair and Translesion Synthesis

Laureti

Lee

Philippin

et al. 2017

Preprint

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The RecBCD complex is a key factor in DNA metabolism. This protein complex harbors a processive nuclease and two helicases activities that give it the ability to process duplex DNA ends. These enzymatic activities make RecBCD a major player in double strand break repair, conjugational recombination and degradation of linear DNA. In this work, we unravel a new role of the RecBCD complex in the processing of DNA single-strand gaps that are generated at DNA replicationblocking lesions. We show that independently of its nuclease or helicase activities, the entire RecBCD complex is required for recombinational repair of the gap and efficient translesion synthesis. Since none of the catalytic functions of RecBCD are required for those processes, we surmise that the complex acts as a structural element that stabilizes the blocked replication fork, allowing efficient DNA damage tolerance. INTRODUCTIONGenomes of all living organisms are constantly damaged by endogenous and exogenous stresses.Despite efficient repair mechanisms, some DNA lesions can escape repair and block the replicative polymerase. In order to bypass these "roadblocks" and complete replication, cells have developed two DNA Damage Tolerance (DDT) pathways identified both in prokaryotes and eukaryotes: 1) Translesion Synthesis (TLS), which employs specialized DNA polymerases able to replicate damaged DNA, with the potential to introduce mutations (1); 2) Damage Avoidance (DA) pathways (also named template switching), which use the information of the sister chromatid to bypass the lesion in a non-mutagenic w a y t h r o u g h h o m o l o g o u s r e c o m b i n a t i o n mechanisms (2, 3). While the TLS pathway has been well characterized in the past few years, little is still known about Damage Avoidance pathways.We have recently developed a genetic tool that enables us to monitor in vivo the exchange of genetic information between sister chromatids (i.e. DA events), following the insertion of a single lesion into the chromosome of Escherichia coli (4). We showed that after encountering a replicationblocking lesion, either on the lagging or the leading strand, the replication fork is able to restart

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RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms

Cited by 38 publications

References 124 publications

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

SbcC-SbcD and ExoI process convergent forks to complete chromosome replication

A non-catalytic role of RecBCD in Homology Directed Gap Repair and Translesion Synthesis

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