Resolution of Converging Replication Forks by RecQ and Topoisomerase III  Catherine Suski, Kenneth J. Marians  Molecular Cell  Volume 30, Issue 6, Pages 779-789 (June 2008) DOI: 10.1016/j.molcel.2008.04.020 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Pathways of Resolution of Replicating Sister DNA Molecules The example shows θ-type replication of a supercoiled DNA template (i). Late replication intermediates (ii) can be resolved by one of two pathways. In pathway A, replication continues through the region of unreplicated parental DNA, generating duplex catenanes for each duplex turn of DNA replicated (iii). Once the gaps in the sister DNA molecules are sealed (iv), these topologically linked sister DNAs can only be decatenated by a type II topoisomerase to give the final product DNA (v). In pathway B, unlinking by a type I topoisomerase is concurrent with replication through the region of unreplicated parental DNA (vi), generating two form II sister DNA molecules (vii) that can then be sealed to give the final product DNA (v). This figure is reproduced from Minden and Marians (1986). Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Resolution of Converging, Stalled Replication Forks by RecQ and Topo III (A) Schematic of LRI preparation and resolution. pBROTB I or II 535-80 plasmid DNA (i) is replicated in the presence of Tus, generating an LRI (ii) where the 3′ OH ends of the nascent leading strands of the converging replication forks are separated by about 130 bp (iv). Treatment of the LRI with RecQ and Topo III yields two gapped, form II sister DNA molecules. (B) Products of the replication reaction in either the absence or presence of Tus. (C) The LRI resolution reaction. Reaction mixtures containing 5 nM RecQ, 8 nM Topo III, and 400 nM SSB, as indicated, were incubated and analyzed as described in the Experimental Procedures. A note about topology of the LRI: if all the LRI synthesized during the replication reaction had formed perfectly, when RecQ unwinds the LRI one would expect to observe a single band at the 13th rung of the ladder of ss catenanes. Such perfect synthesis of the LRI would require that Tus never dissociates from the Ter sites, that the replisome dissociates instantly when it stalls at Tus, and that DNA polymerase I, which is capable of strand-displacement synthesis and is present in the reaction mixture, does not gain access to the 3′ OH ends of the nascent DNA. However, this is not what happens during the 15 min incubation used to accumulate LRI product. Replication of any particular plasmid template takes about 5 s. We have shown previously that stalled replisomes that were formed at and progressed away from oriC in this plasmid replication system in vitro have a half-life of about 4 min (Marians et al., 1998). Thus, if Tus dissociates, the stalled replisomes are able to progress through the parental region between the two Ter sites. This progression, however, is limited by the inability of DNA gyrase, the only topoisomerase in the reaction mixture, to bind ahead of the replication forks to remove positive supercoils. In addition, DNA polymerase I might extend the 3′ OH ends. Consequently, the LRI actually consists of a distribution of almost completely replicated daughter molecules with differing extents of unreplicated parental DNA between the two Ter sites. Unwinding by RecQ in the absence of unlinking by Topo III therefore produces the observed ladders of ss catenanes. As one would expect, the concentrations of Tus, DNA gyrase, and DNA polymerase I in the replication reaction will influence the extent of replication between the Ter sites. The concentrations of these proteins were adjusted to limit this replication as we worked with the system, thus accounting for the different extent of catenation observed in Figures 2–4 compared to Figures 6 and 7. Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 The HRDC Domain of RecQ Is Not Required for LRI Resolution (A) Titration of RecQ in LRI resolution reactions containing 8 nM Topo III and 400 nM SSB. (B) Titration of RecQΔHRDC in LRI resolution reactions containing 8 nM Topo III and 400 nM SSB. (C) Quantification of the experiments shown in (A) and (B). Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Neither Topo I nor Topo IV Can Substitute for Topo III in LRI Resolution (A) Titration of Topo III in LRI resolution reactions containing 5 nM RecQ and 400 nM SSB. (B) Quantification of the experiment shown in (A). (C) Comparison of the activities of Topo I, Topo III, and Topo IV in LRI resolution. LRI resolution reactions contained 5 nM RecQ, 400 nM SSB, 8 nM Topo III, and the indicated concentrations of either Topo I or Topo IV. Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Specificity for RecQ in LRI Resolution (A) LRI resolution reactions contained 8 nM Topo III, 400 nM SSB, and the indicated concentrations of RecQ, Rep, or UvrD. The 2.0 kb fragment is described in the text. (B) LRI resolution reactions contained 8 nM Topo III, 400 nM SSB, and the indicated concentrations of RecQ, PriA, RecG, or RuvAB (reconstituted at a ratio of 5:1, RuvA:RuvB). Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Topo III Stimulates RecQ Unwinding of the LRI LRI resolution reactions contained 400 nM SSB, either no topoisomerase or either 8 nM Topo I or 8 nM Topo III, and the indicated concentrations of RecQ. The fraction of the LRI either unwound or resolved is displayed in the table below the figure of the gel. Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 SSB Mediates Cooperation of RecQ and Topo III in LRI Resolution (A) LRI resolution reactions contained 10 nM RecQ, either no or 6 nM Topo III, as indicated, and either no or 400 nM wild-type SSB, SSBΔC8, or SSB113, as indicated. (B) Effect of variant SSBs on unwinding of the LRI by RecQ. Reactions contained LRI, the indicated concentrations of RecQ, and either no or 400 nM wild-type SSB, SSBΔC8, or SSB113, as indicated. The fraction of the LRI either unwound or resolved is displayed in the table below the figure of the gel. (C) Effect of variant SSBs on Topo III decatenation of unwound LRI. LRI was first unwound by RecQ in a separate reaction containing only 400 nM SSB. The reaction was terminated as described in the Experimental Procedures, extracted with phenol-CHCl3, and the unwound LRI recovered by ethanol precipitation. Unwound LRI was then incubated under standard conditions in reactions containing 7 nM Topo III and either no or 400 nM wild-type SSB, SSBΔC8, or SSB113, as indicated. The fraction of the DNA products as LRI, unwound LRI, or resolved LRI is displayed in the table below the figure of the gel. (D) RecQ and Topo III interact with SSB. Coprecipitation assays were performed as described in the Experimental Procedures. Reaction mixtures contained the indicated proteins. P, pellet; S, supernatant. (E) Neither RecQ nor Topo III interacts with SSBΔC8. Coprecipitation assays were performed as described in the Experimental Procedures. Reaction mixtures contained the indicated proteins. P, pellet; S, supernatant. (F) Topo III, but not RecQ, retains interaction with SSB113. Coprecipitation assays were performed as described in the Experimental Procedures. Reaction mixtures contained the indicated proteins. P, pellet; S, supernatant. Molecular Cell 2008 30, 779-789DOI: (10.1016/j.molcel.2008.04.020) Copyright © 2008 Elsevier Inc. Terms and Conditions