Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Replication forks formed at bacterial origins often encounter template road blocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of th e corrupting template lesion and origin-independent assembly of a new repli some therefore are required for survival of the bacterium. A number of mode ls for replication-fork restart under these conditions posit that nascent s trand 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 fo rks stalled in vitro by the accumulation of excess positive supercoils coul d be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecC, con firming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent stran d regression at stalled replication forks. Furthermore, RecC promoted Holli day junction formation when replication intermediates in which the replisom e had been inactivated were negatively supercoiled, suggesting that under i ntracellular conditions, the action of RecC, or helicases with similar acti vities, is necessary for the catalysis of nascent strand regression.