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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumFormation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiledPeter McGlynn†‡, Robert G.Lloyd†, and Kenneth J.Marians‡§ †Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom; and §Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression. The picture of how DNA replication proceeds around the bacterial chromosome has changed over the last decade as a result of research in many laboratories (1). Even though the two replication forks that form at oriC have a sufficiently high enough inherent processivity to complete replication of the chromosome, it is clear that this is generally not what happens. Instead, the replication forks formed at the origin become inactivated at high frequency as a result of an encounter with roadblocks either in or on the template strands. These roadblocks can take many forms: a nick in one of the template strands, a DNA adduct formed as a result of endogenous damage, secondary structure in the template, and frozen proteins on the DNA. Survival then depends on both correction of the damage and reactivation of DNA replication. Although there is a large body of both genetic and biochemical data informing the mechanisms that act to repair damaged nucleotides in DNA, except in one instance, the mechanisms of replication-fork restart are less well defined. Replication-fork restart after an encounter with a template nick, leading to double-strand break (DSB) generation and detachment from the growing fork of one of the nascent sister duplexes—sometimes termed replication fork collapse (2)—is effected by a marriage of homologous recombination and DNA replication proteins (3). Here, the DSB generated is processed by RecBCD to generate a recombinogenic 3′ single-stranded tail that is used for RecA-catalyzed strand invasion with the intact sister duplex, creating a D loop. This structure is recognized by PriA (4, 5), which then directs the assembly of a new replication fork at the site through the loading of a primosome (6). The strand crossover initiating D loop formation is resolved subsequently. Both the DNA structures formed and the mechanism of replication restart in other cases are less clear. Insight to the problem has been acquired through the examination of the consequences of stalling replication forks in vivo by various means. Placement of Ter sequences outside of the usual configuration at the terminus region of the chromosome generated strains that required RecA and RecBCD for survival if the replication-fork arrest protein Tus also was present (7, 8). The models developed to explain these observations suggested that DSB formation was occurring at the stalled replication fork. Replication-fork restart then presumably could proceed via the pathway discussed above for restart after an encounter with a template nick. Michel and colleagues have studied the consequences of stalling replication forks by interfering with DNA helicase action. They demonstrated an increased frequency of DSB formation in rep recBC mutant strains (9). These researchers argued that the absence of Rep, a 3′ → 5′ DNA helicase (10) known to be able to displace some bound proteins from DNA (11), caused forks to pause more often because of poor clearing of protein obstacles from the template. Interestingly, DSB formation depended on RuvABC (12), the homologous recombination combination branch migration/Holliday junction resolvase machine (13). Thus, this observation suggested that Holliday junctions were forming at stalled replication forks as a result of pairing of the nascent strands and fork regression. RuvAB could either be catalyzing nascent strand regression to form the Holliday junction or be acting subsequent to its formation. Similar observations were made at the nonpermissive temperature in strains carrying conditional-lethal mutations in the replication fork helicase, DnaB (9). Additional processing of the Holliday junction presumably leads to the generation of substrates for replication-fork restart. More recent genetic and biochemical data from McGlynn and Lloyd (14) argues that it is RecG, another branch migration This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: DSB, double-strand break; ERI, early replication intermediate; Topo IV, topoisomerase IV.
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