ATPase/helicase motif mutants of <i>Escherichia coli</i> PriA protein essential for recombination‐dependent DNA replication

Tanaka, Taku; Taniyama, Chika; Arai, Kohei; Masai, Hisao

doi:10.1046/j.1365-2443.2003.00630.x

Cited by 37 publications

(49 citation statements)

References 49 publications

(60 reference statements)

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“…The suggestion that cells lacking RecG and depleted of 39 ssDNA exonucleases suffer damage to their DNA as a result of unscheduled replication fork collisions is consistent with the fact that the problem largely disappears when the helicase activity of PriA is eliminated (Figure 8), especially as PriA helicase mutants reduce SDR (Tanaka et al 2003). Given that DrecG and DrnhA cells have in common a high level of cSDR initiated at R-loops (Kogoma 1997), it is also consistent with the fact that eliminating RNase HI mimics the effect of eliminating RecG (Figure 9).…”

Section: Discussionsupporting

confidence: 67%

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

et al. 2010

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Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 39 flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 39 single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.

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Section: Discussionsupporting

confidence: 67%

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

et al. 2010

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“…Although helicase-deficient PriA proteins retain the ability to assemble a primosome and to complement the DNA repair and growth defects associated with a priA null allele (Zavitz and Marians 1992), they reduce SDR quite substantially (Tanaka et al 2003), a fact consistent with genetic data indicating that the K230R derivative may not be able to initiate replication at D loops . In doing so, we believe they reduce the chromosome pathology arising as a consequence of unscheduled DNA replication (Rudolph et al 2009a,b).…”

Section: Discussionsupporting

confidence: 53%

Promoting and Avoiding Recombination: Contrasting Activities of the Escherichia coli RuvABC Holliday Junction Resolvase and RecG DNA Translocase

et al. 2010

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RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC.

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“…We previously reported that a D-loop like structure strongly stimulated ATPase activity of PriA (29). Similarly, A-fork [3Ј] DNA significantly activated PriA-mediated ATP hydrolysis (Fig.…”

Section: Nuclease Protection Analyses On Thementioning

confidence: 52%

“…Non-structured single-stranded DNA can also activate ATP hydrolysis by PriA in the absence of single-stranded DNAbinding protein (29). This activation is largely dependent on the presence of a 3Ј terminus (Fig.…”

Section: ј Terminus-dependent Bipartite Interaction Of Pria With Anmentioning

confidence: 99%

Escherichia coli PriA Protein, Two Modes of DNA Binding and Activation of ATP Hydrolysis

Tanaka

Mizukoshi

Sasaki

et al. 2007

Journal of Biological Chemistry

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Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3 terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3 terminus recognition. The former mode of binding requires the 3 terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.

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ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination‐dependent DNA replication

Cited by 37 publications

References 49 publications

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

Promoting and Avoiding Recombination: Contrasting Activities of the Escherichia coli RuvABC Holliday Junction Resolvase and RecG DNA Translocase

Escherichia coli PriA Protein, Two Modes of DNA Binding and Activation of ATP Hydrolysis

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