Regression of Replication Forks Stalled by Leading-strand Template Damage

Abstract

Background: Stalled replication forks are foci for genomic instability. Results: Both RecG and RuvAB can regress stalled forks; however, RuvAB completely unwinds the nascent DNA, whereas RuvC cleaves the Holliday junctions formed by RecG. Conclusion: RecG and RuvAB activities are distinct. Significance: Replication fork regression is a major step in processing stalled forks.

Regression of Replication Forks Stalled by Leading-strand Template Damage

“…However, although there was a slight increase in background cleavage by RuvC alone, we did not observe any increase in cleavage products in the presence of RecA. We conclude that RecA, unlike RecG and RuvAB (14), is unable to access the stalled fork in a manner that allows RFR when the replisome and SSB are also present.…”

“…No products were observed ( Fig. 2A), although the control of RecG-RuvC gave the expected cleavage (14).…”

“…Replication Fork Reversal Assays-All reagents and procedures were as detailed in the accompanying article (14) with the following exceptions. To seal Okazaki fragments, DNA polymerase I (New England Biolabs), RNase H, E. coli DNA ligase, and NAD ϩ were added to the replication reaction mixtures to final concentrations of 2 nM (0.0005 units/l), 0.2 nM, 0.08 units/l, and 100 M, respectively.…”

“…RFR reaction mixtures containing 15 l of this preparation of deproteinized stalled forks, 3 M RecA, and 10 nM RuvC, as indicated, were incubated at 37°C for 20 min. Reactions were terminated by the addition of EDTA to 30 M and analyzed by native agarose gel electrophoresis as described (14). The following oligonucleotides were used to prepare the additional templates used in the experiments described in Fig.…”

“…We have used a DNA replication system reconstituted with purified proteins and a DNA template containing specifically located leadingstrand template damage (13) to stall replication forks and examine their susceptibility to regression by these proteins. Our studies with RecG and RuvAB are reported in the accompanying article (14); here, we describe our studies with RecA.…”