Replication Restart in Bacteria

Abstract

In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.KEYWORDS DNA replication, homologous recombination R eplication of a circular bacterial chromosome normally initiates at a unique origin. For bidirectional DNA replication, two replication forks are established that replicate the DNA in opposite directions until they meet in the terminus region. Replication fork progression involves the coordinated action of two complexes, the primosome to open the DNA duplex and synthesize primers and the replisome to catalyze the concerted DNA synthesis of both DNA strands. The two DNA strands at the replication fork are antiparallel, and DNA synthesis occurs only in the 5=-to-3= direction. Therefore, to synthesize both strands in a concerted and semiconservative fashion, one strand is synthesized mainly continuously (the leading strand) and the other is synthesized discontinuously (the lagging strand). The fragments generated on the discontinuous strand are 1 to 2 kb in length and are called Okazaki fragments (OF). In Escherichia coli, the primosome is composed of the DnaB helicase that opens the parental strands and the DnaG primase that interacts transiently with DnaB and synthesizes the RNA primers at the onset of each OF synthesis. DnaB is a hexameric helicase that encircles singlestranded DNA (ssDNA) and translocates on the lagging-strand template in a 5=-to-3= direction. The DNA polymerase III holoenzyme (Pol III HE) synthesizes both nascent DNA strands, and its action is stimulated by interactions with DnaB and with the ssDNA binding protein (SSB) that covers the lagging-strand template (1).The first committed step in the assembly of a replication fork is DnaB loading. In bacteria, two pathways for DnaB loading have been described, a sequence-specific, DnaA-dep...

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria

“…In vitro studies of replisome components led to the model that DNA replication could proceed from origin to terminus without interruption due to the stability of these purified, reconstituted complexes. However, genetic studies had identified the Pri proteins long ago, and through a large body of work, demonstrated that Pri proteins are essential in bacteria, they recognize stalled replication fork structures, and they reassemble the replisome outside of oriC (37). Presumably, if the replisome synthesized DNA largely uninterrupted from oriC to the terminus, replication restart mechanisms would not be essential.…”

Function of a strand-separation pin element in the PriA DNA replication restart helicase

“…tional PriA, and a priA priC double mutation is synthetically lethal due to the essential nature of DNA replication restart (7,9). A transduction experiment was carried out to determine whether the priA⌬␤H allele could be combined with a priC-null allele that is lethal in combination with a priA-null allele.…”

“…The CRR ␤-hairpin also serves a role in PriB interaction with the PriA-DNA complex E. coli have two PriA-mediated DNA replication restart pathways: a PriA-PriB and a PriA-PriC pathway (5,7). Because the priA⌬␤H mutant behaves like a priA-null mutation, it appears that deletion of the tip of the CRR ␤-hairpin has ablated essential PriA functions required in both the PriA-PriB and PriA-PriC pathways.…”

“…In bacteria, these encounters can cause replication fork collapse, leaving abandoned replication forks that are lethal if left unresolved (3,4). Bacteria use conserved "DNA replication restart" pathways to reinitiate DNA replication at prematurely terminated replication sites (5)(6)(7). The PriA DNA helicase is the primary protein that orchestrates replication restart in bacteria.…”

RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway inE.coli

“…This is particularly true when DNA replication encounters DNA lesions, including damaged bases, single and/or double-strand breaks, DNA strand templates bound by proteins etc. [1][2][3][4][5][6][7][8][9][10]. In the last decades, the roles of certain homologous recombination proteins including RecG, RecQ, RuvAB, PriA, PriB, RecA and RecFOR have become increasingly clear [3, [11][12][13][14][15][16][17]].…”