Figure 1.
Model for replication fork reversal in a dnaEts mutant (adapted from [8],[20]).
In the first step (A), the replication fork is arrested by inactivation of dnaE. RuvAB catalyzes the annealing of leading and lagging strand ends, i.e. fork reversal. The reversed fork forms a four-arm structure (Holliday junction, HJ; two alternative representations of this structure are shown, open X and parallel stacked X). RecBC is essential for resetting of the fork, either by RecA-dependent homologous recombination (B–C) or by DNA degradation (B–D). In the absence of RecBCD (E), resolution of the HJ causes chromosome linearization. Continuous lines: parental chromosome. Dashed lines: newly-synthesized strands. Circle: RuvAB. Incised circle: RecBCD. B: Model of RuvAB action at blocked forks. In the first step, a RuvA tetramer binds to the fork and drives the assembly of a RuvB hexamer on the template strands. The translocase action of this RuvB hexamer pulls the leading and lagging strands into the RuvA complex (direction of migration of DNA is indicated by arrows) and results in the formation of a HJ. This HJ is bound by a second RuvB hexamer forming a bona fide branch migration complex (direction of translocation of DNA is indicated by arrows, it is unclear at present whether the active form of the branch migration complex in vivo carries one or, as drawn here, two tetramers of RuvA). HJ resolution by RuvC results in a cleaved replication fork.
Table 1.
ruvA mutant alleles deficient for RFR.
Figure 2.
Positions of ruvAz3 and ruvA87 mutations.
The mutations in RuvAz3 and RuvAz87 on the primary sequence (A) are shown in blue (H29R K129E F140S) and in yellow (N79D N100D), respectively. Full and dashed lines indicate the positions of the two helix-turn-helix in domains II and of the disordered segment that separates domains II and III, respectively [33]The positions of the three restriction sites used to separate the mutations in the original ruvAz60 allele are shown above the sequence. Domain I (1 to 64), II (65–140) and III (156–203) are not indicated. The mutations are also shown as blue (RuvAz3) or yellow (RuvAz87) spheres in a ribbon view of the 3D structure of RuvA, viewed at the DNA-binding face (B) and a perpendicular side view of this (C).
Table 2.
Three mutations in pGB-ruvAz60 are necessary and sufficient to inactivate RFR.
Table 3.
Increasing the amount of RuvB restores RFR in ruvAz mutants.
Figure 3.
ruvAz3 and ruvA87 suppress the recombination defect of a ruvA100 mutant.
(A) Exponentially growing JJC 2971 (ruvA100) cells containing different plasmids were treated with 2 µg/ml mitomycin C for 90 min, plated on LB-spectinomycin and incubated over-night. Ratios of colony forming units (cfu) in treated vs untreated cultures are shown. (B) Exponentially growing cells were mixed with a His+ Hfr donor for 25 min, plated on chloramphenicol minimal medium devoid of histidine and incubated for 48 hours. Ratios of His+ vs total recipient cfu are shown. Recipient RecG+ JJC2971 (ruvA100), recipient recG− JJC3207 (ruvA100 recG::kanR). (C) Appropriate dilutions of exponentially growing JJC 2971 (ruvA100) cells containing different plasmids were plated on LB-spectinomycin, UV-irradiated, and incubated over-night. Ratios of cfu on irradiated vs non-irradiated plates were calculated. Average of at least three values and standard deviations are shown. Diamonds: pGB2, squares: pGB-RuvA+, circles: pGB-ruvAz3, triangles: pGB-ruvAz87. (D) Same experiments with JJC3375 (ruvA100 recR), symbols are as in panel C. (E) Same experiments with JJC3207 (ruvA100 recG), symbols are as in panel C. (F) same experiments with JJC2761 (ΔruvABC rus-1), closed symbols are as in panel C, dashed line-open circles: pGB-ruvAz3-RuvB+, dashed line-open triangles: pGB-ruvAz87-RuvB+.
Figure 4.
RuvAz3 and RuvAz87 proteins are deficient for octamerisation on HJs and slightly affected for HJ branch migration.
(A) binding assay: Fluorescence-labeled junction ×12 (∼4 ng) was incubated in the presence of 5 mM EDTA with varying amounts of wild-type (wt) RuvA, RuvAz3 or RuvAz87, as indicated. Binding curves were obtained by quantification using Li-cor Biosciences ODYSSEY infrared imaging system. Squares: RuvA, circles: RuvAz3, triangles: RuvAz87. (B) as in A but in the presence of 3 mM Mg2+ (in the absence of EDTA). (C) branch migration assay: Reaction mixtures containing ∼4 ng labeled synthetic ×12 junctions were incubated withf 250 nM RuvB and various amounts of wild-type or mutant RuvA, as indicated. Lane 2 is the substrate only. Gels were quantified as in A. Symbols are as in A.
Figure 5.
RuvAz3 is more deficient for the stimulation of RuvB helicase activity than RuvAz87.
DNA helicase substrate consisting of fluorescence-labeled 52mer oligonucleotide annealed to ϕX174 single-stranded DNA (4 ng) was incubated with 250 nM RuvB and various amounts of wild-type or mutant RuvA, as indicated. Gels were quantified as in Figure 4. Symbols are as in Figure 4.
Figure 6.
RuvAz3 and RuvAz87 are deficient for fork binding and fork unwinding activity.
(A) binding assay: Fluorescence-labeled F2 forks (∼4–5 ng) in which the three DNA arms are double-stranded, were incubated in the presence of 5 mM EDTA with varying amounts of wild-type RuvA or mutant RuvAz3, RuvAz87, as indicated. No binding was detected with mutant RuvA proteins and only the highest concentrations of protein used are shown. (B) as in A but with F1, in which one of the three arms is single-stranded. (C) fork unwinding assay: Reaction mixtures containing ∼5 ng labeled synthetic F2 forks were incubated with 250 nM RuvB and various amounts of wild-type or mutant RuvA, as indicated. The last two lanes contain the controls shown schematically on the right: a fork substrate with two single-stranded daughter arms and the labeled oligonucleotide. Acting on F2, shown schematically on the left, RuvB binds the two arms that mimic the daughter chromosomes and RuvAB unwinds the arm that mimics the template strands. Gels were quantified as in Figure 4. Symbols are as in Figure 4.