Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator.

Abstract:

:The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage phiX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriA(Bs), we constructed priA(Bs) mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMbeta1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMbeta1 are strongly affected in the mutants. The purified PriA(Bs) protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSB(Bs)). PriA(Bs) also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriA(Bs) recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriA(Bs) also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSB(Bs), unless the ssiA sequence is present on the ssDNA substrate. Finally, PriA(Bs) is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriA(Ec) and PriA(Bs). However, PriA(Bs) does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Polard P,Marsin S,McGovern S,Velten M,Wigley DB,Ehrlich SD,Bruand C

doi

10.1093/nar/30.7.1593

keywords:

subject

Has Abstract

pub_date

2002-04-01 00:00:00

pages

1593-605

issue

7

eissn

0305-1048

issn

1362-4962

journal_volume

30

pub_type

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