Late steps in genetic recombination: branch migration and Holliday junction resolution by RuvA, RuvB, and RuvC proteins.
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Bacterial Proteins↗
Biomedical subjects
S C West
Publications and source records attributed to S C West.
ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli.
The RuvA and RuvB proteins of E. coli, which are induced as part of the cellular response to DNA damage, act together to promote the branch migration of Holliday junctions. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction stimulates the rate of strand exchange and the formation of hetero-duplex DNA. Stimulation does not occur via interaction with RecA; instead, RuvA and RuvB act directly upon recombination intermediates (Holliday junctions) made by RecA. We show that RuvAB-mediated branch migration requires ATP and can bypass UV-induced DNA lesions. At high RuvB concentrations, the requirement for RuvA is overcome, indicating that the RuvB ATPase provides the motor force for branch migration. RuvA protein provides specificity by binding to the Holliday junction, thereby reducing the requirement for RuvB by 50-fold. The newly discovered biochemical properties of RuvA, RuvB, and RuvC are incorporated into a model for the postreplicational repair of DNA following UV irradiation.
Adenosine Triphosphate↗
Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.
The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.
Adenosine Triphosphate↗
Unusual stability of recombination intermediates made by Escherichia coli RecA protein.
The structure and stability of recombination intermediates made by RecA protein have been investigated following deproteinization. The intermediates consist of two duplex DNA molecules connected by a junction, as visualized by electron microscopy. Although we expected the structures to be highly unstable due to branch migration of the junction, this was not the case. Instead, we found that the intermediates were stable at 37 degrees C. At 56 degrees C, greater than 60% of the intermediates remained after 6 h of incubation. Only at higher temperatures was significant branch migration observed. This unexpected stability suggests that the formation of extensive lengths of heteroduplex DNA in Escherichia coli is likely to require the continued action of proteins, and does not occur via spontaneous branch migration. We show that heteroduplex DNA may be formed in vitro by ATP-dependent strand exchange catalysed by RecA protein or by the RuvA and RuvB proteins of E. coli.
Bacterial Proteins↗
Purification and properties of the RuvA and RuvB proteins of Escherichia coli.
The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M(r) ca. 100,000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and beta-mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M(r) ca. 85,000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.
Bacterial Proteins↗
Biological roles of the Escherichia coli RuvA, RuvB and RuvC proteins revealed.
In Escherichia coli, the ruvA, ruvB and ruvC gene products are required for genetic recombination and the recombinational repair of DNA damage. New studies suggest that these three proteins function late in recombination and process Holliday junctions made by RecA protein-mediated strand exchange. In vitro, RuvA protein binds a Holliday junction with high affinity and, together with RuvB (an ATPase), promotes ATP-dependent branch migration of the junction leading to the formation of heteroduplex DNA. The third protein, RuvC, which acts independently of RuvA and RuvB, resolves recombination intermediates by specific endonucleolytic cleavage of the Holliday junction.
Adenosine Triphosphate↗
Enzymes and molecular mechanisms of genetic recombination.
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Animals↗
Action of RecBCD enzyme on Holliday structures made by RecA.
In vitro, Escherichia coli RecA protein acts upon gapped and partially homologous linear duplex DNA to generate recombination products linked by Holliday junctions. When strand exchange reactions are supplemented with purified RecBCD enzyme, we observe the formation of products that resemble "patch" recombinants. The formation of "splice" recombinant products was not observed. The individual subunits, RecB, RecC, or RecD, had no effect on RecA protein-mediated strand exchange nor on the Holliday junctions formed in the reaction. Analysis of the way in which patch products arise indicates exonucleolytic digestion of the linear arms of the recombination intermediates (alpha-structures) by RecBCD enzyme. We find no evidence for specific resolution events at the site of the Holliday junction by RecBCD enzyme using these DNA substrates.
Crossing Over, Genetic↗
Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.
In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.
Bacterial Proteins↗
Three-stranded DNA helices as intermediates in genetic recombination.
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DNA↗
Effects of structured cooperative contact on changing negative attitudes toward stigmatized social groups.
The contact hypothesis predicts that cooperative interaction with members of a disliked group results in increased liking for those members and generalizes to more positive attitudes toward the group. The authors sought to provide evidence consistent with the hypothesis that contact affects attitude in part by eliciting a more positive portrait of the typical group member. Undergraduates participated in a 1-hr dyadic learning session (scripted cooperative learning, jigsaw cooperative learning, or individual study) with a confederate portrayed as a former mental patient. Students initially expected the confederate to display traits similar to those of a typical former mental patient. After the sessions, initially prejudiced students in the 2 cooperative conditions described the typical mental patient more positively and adopted more positive attitudes and wider latitudes of acceptance toward the group. Connections between intergroup attitudes and impression formation are discussed.
Attitude↗
T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein.
T7 endonuclease I is known to bind and cleave four-way junctions in DNA. Since these junctions serve as analogues of Holliday junctions that arise during genetic recombination, we have investigated the action of T7 endonuclease I on recombination intermediates containing Holliday junctions. We find that addition of T7 endonuclease I to strand exchange reactions catalysed by RecA protein of Escherichia coli leads to the formation of duplex products that correspond to 'patch' and 'splice' type recombinants. Resolution of the recombination intermediates occurs by the introduction of nicks at the site of the Holliday junction. The recombinant molecules contain 5'-phosphate and 3'-hydroxyl termini which may be ligated to restore the integrity of the DNA.
DNA, Bacterial↗
Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.
T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding complex between T7 endonuclease I and a synthetic Holliday junction analog has been probed with hydroxyl radicals. The results indicate that the nuclease binds all four strands about the junction point.
Base Sequence↗
Underwinding of DNA associated with duplex-duplex pairing by RecA protein.
Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.
Bacteriophage phi X 174↗
Interaction of a four-way junction in DNA with T4 endonuclease VII.
The binding of a synthetic four-way junction in DNA by T4 endonuclease VII has been studied using gel retardation and footprint analysis. Two specific protein-DNA complexes have been observed, but only one is stable in the presence of moderate concentrations of salt. The footprint of T4 endonuclease VII in the salt-resistant complex has been probed using hydroxyl radicals generated by the reaction of iron(II)/EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern indicates protection of approximately 5 residues in two strands that are diametrically opposed across the junction point.
Base Sequence↗
Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions.
Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.
Base Sequence↗
Enzymatic formation and resolution of Holliday junctions in vitro.
E. coli RecA protein promotes homologous pairing and reciprocal strand exchange reactions between duplex DNA molecules in vitro. Reaction intermediates contain Holliday junctions that are driven along the DNA at a maximal rate approaching 1000 bases per minute. T4 endonuclease VII cleaves Holliday junctions in vitro, and its inclusion in RecA-mediated reactions leads to the rapid formation of heteroduplex products. Product analysis indicates patch and splice recombinant molecules similar to those expected from in vivo recombination events. The combined formation and resolution of Holliday junctions has led us to propose a model for resolution based on the structure of RecA-DNA helices. One feature of this model is that resolution, which gives rise to the two types of recombinant product, may occur without need for isomerization of the junction.
Bacteriophage phi X 174↗
Processing of recombination intermediates in vitro.
Genetic recombination involves the exchange of genetic material between chromosomes to produce new assortments of alleles. As such, it affects one of the most fundamental and important components of heredity--the genome itself. To understand the molecular basis of recombination, efforts have been directed to try to determine how simple organisms recombine their DNA. One approach involves the development of in vitro systems in which recombination reactions can be studied using purified enzymes. Detailed studies of these systems, using enzymes isolated from bacteria and bacterial viruses, indicate the formation of unique protein-DNA complexes. The structure of the DNA within these complexes has important consequences for the subsequent formation of recombinant products.
Base Sequence↗