Dissecting the functional role of PriA protein‐catalysed primosome assembly in <i>Escherichia coli</i> DNA replication

Zavitz, Kenton H.; Marians, Kenneth J.

doi:10.1111/j.1365-2958.1991.tb01846.x

Cited by 42 publications

(21 citation statements)

References 35 publications

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“…We suggest that the 3Ј-5Ј helicase activity of UvrD helps maintain this single-stranded DNA region free of secondary structure and that in the absence of UvrD, synthesis of some fraction of Okazaki fragments by DNA polymerase III is impeded so that a chronic, low-level SOS-inducing signal is generated by the unreplicated DNA segments on the lagging strand. A similar function was in fact postulated earlier for PriA (29); however, more recent findings have established that the preeminent role of this protein is in the reassembly of collapsed replication forks (7,18,58) and that loss of its helicase activity is not sufficient for generation of an SOS-inducing signal in vivo (18,48).…”

Section: Discussionmentioning

confidence: 99%

lon Incompatibility Associated with Mutations Causing SOS Induction: Null uvrD Alleles Induce an SOS Response in Escherichia coli

2000

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The uvrD gene in Escherichia coli encodes a 720-amino-acid 3-5 DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind ؊ ), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.DNA helicases are enzymes involved in a variety of processes such as DNA replication, repair, and recombination, and at least 12 have been identified in Escherichia coli (29,30). Of these, only DnaB helicase (with 5Ј-3Ј polarity) is essential for cell viability, being associated with movement of the chromosome replication fork (27). Three other helicases, Rep, PriA, and UvrD, each with 3Ј-5Ј polarity, have also been suggested to participate in chromosome replication, but their roles are not as well established.The UvrD helicase (also called helicase II) is the product of the uvrD gene and is a polypeptide of 720 amino acids. UvrD is required in the pathways of both methyl-directed mismatch repair (34, 35) and UvrABC-mediated excision repair (46, 47), and uvrD mutants exhibit a spontaneous mutator phenotype as well as increased sensitivity to UV irradiation. Genetic evidence has implicated UvrD in homologous recombination (21,23,26,32). The suggestion has also been made that UvrD participates in DNA replication, based on (i) experiments with cell-free systems (17, 22), (ii) the observation that uvrD null mutations are incompatible with rep (encoding the Rep helicase) or polA (encoding DNA polymerase I) mutations (56), and (iii) identification of dominant-lethal uvrD mutations (8,59).In the present study, we discovered that lon (encoding the Lon protease) represents a third locus, mutations in which are incompatible with uvrD null alleles. Chronic induction of the SOS response (reviewed in references 23 and 55) in the uvrD null strains was shown to be the basis for l...

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Section: Discussionmentioning

confidence: 99%

lon Incompatibility Associated with Mutations Causing SOS Induction: Null uvrD Alleles Induce an SOS Response in Escherichia coli

2000

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“…Leading-strand synthesis creates a D-loop and exposes a PAS site for PriA in the displaced strand. PriA is also a DNA-dependent ATPase and after binding at PAS can translocate along DNA in the 3Ј-5Ј direction, using the energy from ATP hydrolysis, unwinding any duplex DNA that it encounters (16,53).…”

Section: Discussionmentioning

confidence: 99%

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12

1996

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The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA ؉ plasmids increase substantially the recombination-and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3-5 DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.

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“…This mutant gene encodes PriA K230R, a PriA protein that is no longer a DNA helicase but can still assemble a primosome (54). To account for the observed induction of SOS in the absence of exogenous DNA-damaging agents, we proposed that PriA primosome assembly activity was required to restart replication forks that had stalled because of encountering endogenous DNA damage (37,53). The mechanism of replication fork assembly emerged as a result of subsequent investigation of the phenotypes of priA mutants.…”

Section: Genetics and Modelsmentioning

confidence: 99%

Role of PriA in Replication Fork Reactivation in Escherichia coli

2000

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2As a result of the work of many laboratories, a new paradigm describing the manner by which bacteria respond to repair DNA damage has emerged. This paradigm holds that under any growth condition, essentially all replication forks formed at oriC encounter DNA damage and either stall or collapse before they can complete synthesis of the genome. Maintenance of cell viability therefore requires both correction of the DNA lesion via the action of the DNA repair enzymes and replication fork restart via the combined action of the DNA recombination and replication enzymes. A proposal has been advanced to distinguish this pathway, which operates as a housekeeping function in the absence of exogenous insult to the cell and is likely to be inherently nonmutagenic, from the SOS response, which is induced by exogenous DNA damage and includes error-prone repair, that it be named CPR for coordinated processing of damaged replication forks (M. M.

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Dissecting the functional role of PriA protein‐catalysed primosome assembly in Escherichia coli DNA replication

Cited by 42 publications

References 35 publications

lon Incompatibility Associated with Mutations Causing SOS Induction: Null uvrD Alleles Induce an SOS Response in Escherichia coli

lon Incompatibility Associated with Mutations Causing SOS Induction: Null uvrD Alleles Induce an SOS Response in Escherichia coli

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12

Role of PriA in Replication Fork Reactivation in Escherichia coli

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