Cellular Characterization of the Primosome and Rep Helicase in Processing and Restoration of Replication following Arrest by UV-Induced DNA Damage in Escherichia coli

Abstract

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion. P riA, PriB, and PriC were originally identified as proteins required for replication of single-strand X174 phage DNA in vitro and in vivo (70,71). In vitro, the proteins function as a complex that is required for processive priming to occur behind the replicative helicase, DnaB (1, 2). PriA initially binds a hairpin structure on the X174 chromosome, followed by PriB, DnaT, and PriC. The resulting complex then recruits DnaC, which loads the DnaB helicase onto the chromosome. The DnaG primase is then able to associate with DnaB to synthesize RNA primers. While DnaG and DnaB are sufficient for primer synthesis on X174 DNA (1), specific and processive priming of singlestranded DNA binding protein-coated phage DNA requires PriA (2). In vivo, conversion of X174 from its plus-strand form to its minus-strand replication intermediate requires PriA and other Escherichia coli host proteins (40). E. coli strains lacking PriA have reduced viability, growth rates, and culture densities relative to wild-type cells (36). priA mutants are also constitutively induced for the SOS response, and cells lacking PriA produce filaments extensively (49). Taken together, these observations led early researchers to propose that the primosomal proteins promote efficient priming for Okazaki fragments during lagging-strand replication (35,38).rep was originally identified as a mutant that was unable to support replication of DNA of several double-stranded phage, including X174 (19,20,33,56,62). rep was subsequently shown to encode a DNA helicase that tracks on the leading-strand template and is essential to reconstitute replication of double-stranded phage in vitro (55). rep mutants ...

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