Essential Role for an Isoform of Escherichia coli Translation Initiation Factor IF2 in Repair of Two-Ended DNA Double-Strand Breaks

Mallikarjun, Jillella; Gowrishankar, J.

doi:10.1128/jb.00571-21

Cited by 9 publications

(10 citation statements)

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“…In the dnaA + recD + control strain (Fig. 7 i), copy number reductions were observed in the vicinity of both I-SceI cleavage sites II and V, but its magnitude was apparently less than that described in the previous studies for a single site of cleavage (42, 55); it is possible that either cleavage by I-SceI, or resection by RecBCD, becomes ratelimiting with more than one I-SceI site on the chromosome. In the dnaA + ΔrecD derivative (Fig.…”

Section: Resultsmentioning

confidence: 64%

“…S2B, compare relative sizes of blue ( recA + ) and white (Δ recA ) colonies in different panels]. This serves to exclude occurrence of spontaneous DSBs in the Δ recD Δ recJ mutant, since a recA mutant cell is killed with even one DSB on the chromosome (12, 13, 42, 53, 86). The triple mutant recD recJ recA of Salmonella typhimurium is also viable (84).…”

Section: Resultsmentioning

confidence: 99%

“…Genome-wide DNA copy number determinations. Copy number determinations of different genomic regions were performed by whole-genome sequencing (WGS) on phenol-chloroform extracted DNA preparations, essentially as described (29,42). WGS read data of 200-fold or greater coverage were generated with a paired-end sequencing strategy on an Illumina HiSeq platform.…”

Section: Methodsmentioning

confidence: 99%

“…Since we had identified that ∆dnaA lethality can be rescued in a ∆recD strain also by imposition of two-ended DSBs at two or more discrete sites through I-SceI cleavage (in presence of ∆tus and rpoB*35 mutations), we determined by WGS the DNA copy number distributions in four of these ∆dnaA-suppressed derivatives: three bearing different combinations of dual I-SceI sites (of which site II near dusA was common and the second numbered site I, III, or V was near yihQ, yaiZ, and nadB, respectively), and one with three I-SceI sites (II, IV and V). All cultures were grown in continuous presence of Ara since this was a requirement for their viability.As controls, dnaA + derivatives of the recD + and ∆recD strains each carrying two I-SceI cleavage sites (II and V) were used in the WGS experiments after growth in continuous presence of Ara (both were also ∆tus and rpoB*35).Previous studies have shown that sustained cleavage in the wild-type strain at a single chromosomal I-SceI site results in attainment of an equilibrium between DNA resection on the one hand and DNA repair on the other, such that an asymmetric V-shaped pattern of reduction in DNA copy number is generated around the cleavage site; this dip in copy number extends to approximately 100 kb towards oriC and 200 kb towards Ter(42,55)…”

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confidence: 95%

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Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Goswami

Gowrishankar

2022

Preprint

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Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation has been that to obtain mutants which survive loss of DnaA. Here we show that a Capital Greek DeltarecD mutation, associated with loss of RecBCD's double strand-DNA exonuclease V activity, provokes abnormal replication and rescues Capital Greek DeltadnaA lethality in two situations: (i) in absence of 5'-3' single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. With two-ended DSBs in a Capital Greek DeltarecD strain, Capital Greek DeltadnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in Capital Greek DeltadnaA-rescued cells lead us to propose a model that exonuclease V-mediated DNA resection activity is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse; such mergers occur during normal replication at the chromosomal terminus region, as well as during repair of two-ended DSBs following "ends-in" replication. Resection mechanisms may also be important in eukaryotes to prevent DNA over-replication at sites of fork merger.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 95%

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Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Goswami

Gowrishankar

2022

Preprint

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“…Copy number determinations of different genomic regions were performed by whole-genome sequencing (WGS) on phenol-chloroform extracted DNA preparations, essentially as described (36,53). WGS read data of 200-fold or greater coverage were generated with a paired-end sequencing strategy on an Illumina HiSeq platform.…”

Section: Genome-wide Dna Copy Number Determinationsmentioning

confidence: 99%

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Goswami

Gowrishankar

2022

Nucleic Acids Research

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Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here, we show that a ΔrecD mutation, associated with attenuation of RecBCD’s DNA double strand end-resection activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5′-3′ single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. One-ended DSBs in the ΔrecD mutant did not rescue ΔdnaA lethality. With two-ended DSBs in the ΔrecD strain, ΔdnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in ΔdnaA-rescued cells lead us to propose a model that nuclease-mediated DNA resection activity of RecBCD is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse, as may be expected to occur during normal replication at the chromosomal terminus region or during repair of two-ended DSBs following ‘ends-in’ replication.

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Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

McKenzie

Henry

Myers

et al. 2022

G3 Genes|Genomes|Genetics

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Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break (DSB) accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in DSB homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

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Essential Role for an Isoform of Escherichia coli Translation Initiation Factor IF2 in Repair of Two-Ended DNA Double-Strand Breaks

Abstract: Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli , DSBs are repaired by RecA- and RecBCD-mediated homologous recombination (HR).

Cited by 9 publications

References 70 publications

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

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