Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication.

Abstract

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS‐induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174‐type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild‐type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA‐dependent phi X174‐type primosome in these DnaA/oriC‐independent pathways of chromosome replication. Whereas ColE1‐type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA‐dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Binding of PriA Protein Requires Cooperation of the N-terminal D-loop/Arrested-fork Binding and C-terminal Helicase Domains

“…Oligonucleotides used in this study Measurement of iSDR-iSDR was measured as described previously (3,9,32). Briefly, cells, grown in M9 medium at 37°C overnight, were diluted to A 600 0.5 and were transferred to M9 medium without thymine.…”

“…priA null cells generally exhibit low viability (5,6), and do not form colonies on rich broth medium (3). They are hypersensitive to DNA damaging agents such as UV (4,5,7), and a subpopulation exhibits extremely filamentous morphology (5,6,8).…”

“…Whereas iSDR does not require transcription, cSDR completely depends on ongoing transcription. It was proposed that iSDR and cSDR are initiated from D-loop and R-loop structures, respectively, on the chromosome (3,13,14).…”

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

“…It has been suggested that RecG also promotes replication of damaged DNA Lloyd 2000, 2002a,b), but this has proven more contentious (Donaldson et al 2004;Michel et al 2007). What is not in doubt is the ability of RecG to limit the incidence of DnaAindependent SDR, which is mediated via PriAdependent loading of DnaB and subsequent replisome assembly at D loops and R loops (Asai and Kogoma 1994a,b;Masai et al 1994;Hong et al 1995). Without RecG to dissociate these branched structures (Vincent et al 1996;Fukuoh et al 1997), replication may initiate wherever they arise, rather than being restricted to DnaA-dependent events at oriC.…”

Identification of Proteins Required for Repair of Double-Strand Chromosome Breaks, a Predisposing Factor in Breast Cancer

“…The construction of knockout strains (priA1 and priA2) in Escherichia coli has revealed severe and complex phenotypic effects, which include reduced viability (=10 2 -fold), slow growth, aspects of a constantly induced SOS response, UV-hypersensitivity and reduced doublestrand break repair (Lee and Kornberg, 1991;Nurse era/., 1991;Masai et al, 1994). PriA is also required for recombination-dependent forms of chromosomal replication as well as for normal levels of assimilation of genetic markers by homologous recombination (Masai et al, 1994;Kogoma era/., 1996). These observations suggest that PriA is the factor that couples replication and recombination in the cell.…”