Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.     

RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.

The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.     

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.     

Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer

The Escherichia coli RuvA and RuvB protein complex promotes branch migration of Holliday junctions during recombinational repair and homologous recombination and at stalled replication forks. The RuvB protein belongs to the AAA(+) (ATPase associated with various cellular activities) ATPase family and forms a hexameric ring in an ATP-dependent manner. Studies on the oligomeric AAA(+) class ATPases suggest that a conserved arginine residue is located in close proximity to the ATPase site of the adjacent subunit and plays an essential role during ATP hydrolysis. This study presents direct evidence that Arg-174 of RuvB allosterically stimulates the ATPase of the adjacent subunit in a RuvB hexamer. RuvBR174A shows a dominant negative phenotype for DNA repair in vivo and inhibits the branch migration catalyzed by wild-type RuvB. A dominant negative phenotype was also observed with RuvBK68A (Walker A mutation). RuvB K68A-R174A double mutant demonstrates a more severe dominant negative effect than the single mutants RuvB K68A or R174A. Moreover, although RuvB K68A and R174A are totally defective in ATPase activity, ATPase activity is restored when these two mutant proteins are mixed at a 1:1 ratio. These results suggest that each of the two mutants has distinct functional defects and that restoration of the ATPase activity is brought by complementary interaction between the mutant subunits in the heterohexamers. This study demonstrates that R174 plays an intermolecular catalytic role during ATP hydrolysis by RuvB. This role may be a general feature of the oligomeric AAA/AAA(+) ATPases.     

Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer

In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-A resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.     

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.     

Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvA-RuvB protein complex

The Escherichia coli RuvA-RuvB complex promotes branch migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA-RuvB promotes branch migration by driving helical rotation of the DNA. To clarify the RuvA-RuvB-mediated branch migration mechanism in more detail, we observed DNA rotation during Holliday junction branch migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA-RuvB-mediated branch migration at various ATP concentrations. The data provided a K(m) value of 65 microM and a V(max) value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA-RuvB-mediated branch migration, but also opens the possibility of elucidating the branch migration mechanism in detail.     

Escherichia coli RecQ protein is a DNA helicase.

The Escherichia coli recQ gene, a member of the RecF recombination gene family, was set in an overexpression plasmid, and its product was purified to near-homogeneity. The purified RecQ protein exhibited a DNA-dependent ATPase and a helicase activity. Without DNA, no ATPase activity was detected. The capacity as ATPase cofactor varied with the type of DNA in the following order: circular single strand greater than linear single strand much greater than circular or linear duplex. As a helicase, RecQ protein displaced an annealed 71-base or 143-base single-stranded fragment from circular or linear phage M13 DNA, and the direction of unwinding seemed to be 3'----5' with respect to the DNA single strand to which the enzyme supposedly bound. Furthermore, the protein could unwind 143-base-pair blunt-ended duplex DNA at a higher enzyme concentration. It is concluded that RecQ protein is a previously unreported helicase, which might possibly serve to generate single-stranded tails for a strand transfer reaction in the process of recombination.     

Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins.     

DNA-dependent protein kinase: DNA binding and activation in the absence of Ku

In mammalian cells, double-strand break repair and V(D)J recombination require DNA-dependent protein kinase (DNA-PK), a serine/threonine kinase that is activated by DNA. DNA-PK consists of a 460-kDa subunit (p460) that contains a putative kinase domain and a heterodimeric subunit (Ku) that binds to double-stranded DNA ends. Previous reports suggested that the activation of DNA-PK requires the binding of Ku to DNA. To investigate this further, p460 and Ku were purified separately to homogeneity. Surprisingly, p460 was capable of binding to DNA in the absence of Ku. The binding of p460 to double-stranded DNA ends was salt-labile and could be disrupted by single-stranded or supercoiled DNA, properties distinct from the binding of Ku to DNA. Under low salt conditions, which permitted the binding of p460 to DNA ends, the kinase was activated. Under higher salt conditions, which inhibited the binding of p460, activation of the kinase required the addition of Ku. Significantly, when the length of DNA decreased to 22 bp, Ku competed with p460 for DNA binding and inhibited kinase activity. These data demonstrate that p460 is a self-contained kinase that is activated by direct interaction with double-stranded DNA and that the role of Ku is to stabilize the binding of p460 to DNA ends.     

Homologous pairing in genetic recombination: complexes of recA protein and DNA.

recA protein, which is essential for general genetic recombination in Escherichia coli, promotes the homologous pairing of single-stranded DNA with double-stranded DNA to form a D loop. The amount of recA protein required for the reaction was directly proportional to the amount of single stranded DNA and was unaffected by similar variations in the amount of double-stranded DNA. The ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which was not rapidly hydrolyzed by recA protein, blocked the formation of D loops but promoted the formation of stable complexes of recA protein and single-stranded DNA. These complexes, in turn, bound homologous or heterologous double-stranded DNA and partially unwound it. Because ATP gamma S competitively inhibited the ATPase activity of recA protein (Km/Ki approximately 300), we infer that ATP gamma S binds at a site that overlaps the site for ATP and that the functional complexes formed in the presence of the analog probably represent partial steps in the overall reaction. If the complexes formed in the presence of ATP gamma S reflect natural intermediates in the formation of D loops, recA protein must promote homologous pairing either by moving juxtaposed single-stranded and double-stranded DNA relative to one another or by forming and dissociating complexes reiteratively until a homologous match occurs.     

Recognition of chemical carcinogen-modified DNA by a DNA-binding protein.

Using a filter binding assay, we have detected and partially purified a protein from human placenta that has a high affinity for N-acetoxy-2-acetylaminofluorene-modified double-stranded DNA (AAF-[3H]DNA) of bacteriophage T7. This protein has been partially purified from a 1 M NaCl extract of a crude nuclear fraction by a combination of ion-exchange and nucleic acid affinity chromatography. With AAF-[3H]DNA as the substrate, the binding reaction reached equlibrium within 1 hr at 4 degrees C, and the extent of binding ws proportional to the amount of protein added. Complex formation was dependent on both pH and salt concentration and was unaffected by the presence of sulfhydryl-blocking agents. The purest protein fraction also recognizes DNA modified with methylmethane-sulfonate or methylnitrosourea. It shows little or no recognition of single-stranded DNA, double-stranded DNA, supercoiled bacteriophage phiX174 DNA, partially depurinated DNA, glucosylated bacteriophage T4DNA, or UV-irradiated DNA. No endo- or exonuclease activity, DNA polymerase activity, or glucosylase activity for AAF-DNA was detectable in the preparation.     

Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca(2+) greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca(2+) exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg(2+), the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca(2+) maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein.     

Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA

Bacillus subtilis RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) ( approximately 10- and approximately 40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the epsilon epistatic group (namely ruvA, recD, and recU). In the presence of Mg(2+), RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.     

Purification and properties of the uvrA protein from Escherichia coli.

The uvrA+ gene product from Escherichia coli was purified to apparent homogeneity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 molecular weight DNA-binding protein having higher affinity for single-stranded than double-stranded DNA. It does not introduce single-strand breaks or alkali-labile bonds in native or UV-irradiated DNA, but it catalyzes hydrolysis of ATP to ADP and Pi. The ATPase activity is not DNA dependent and has a Km of 0.23 mM, which corresponds to the Km for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+, and uvrC+ gene products. ADP and adenosine 5'-[gamma-thio]triphosphate both inhibit the uvrA ATPase as well as the uvrABC endonuclease and also prevent specific binding of the uvrA proteins to UV-irradiated DNA. These results indicate that both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity and that the ATP requirement of the endonuclease reaction is determined by uvrA ATPase.     

Identification of a specific inhibitor for DNA ligase I in human cells.

A protein inhibitor for human DNA ligase I has recently been identified. It was copurified with a fraction of the enzymes from HeLa cells through several steps of chromatography. The inhibitor was first identified by the absence of ligation activity of the associated enzyme, while it retained the ability to form the ligase-[32P]AMP adducts. The inhibitor was eluted as a single peak at approximately 0.25-0.30 M NaCl from a Mono S column. It inhibited the ligation of both double-stranded and single-stranded breaks by purified DNA ligase I but not by T4 DNA ligase and DNA ligase II. Subsequent gel-filtration chromatography indicated that this inhibitor, with a molecular mass of 55-75 kDa, could form a complex with DNA ligase I and inhibited the DNA ligation activity. Rechromatography of the ligase I-inhibitor complex in high-salt conditions resulted in the dissociation of the complex and the restoration of enzyme activity, indicating that the physical interaction of inhibitor with DNA ligase I is one of the mechanisms of inhibition. These data indicate that this protein inhibitor for DNA ligase I may play a specific role in regulating DNA ligation during replication, repair, or recombination.     

Gin-mediated DNA inversion: product structure and the mechanism of strand exchange.

Inversion of the G loop of bacteriophage Mu requires the phage-encoded Gin protein and a host factor. The topological changes in a supercoiled DNA substrate generated by the two purified proteins were analyzed. More than 99% of the inversion products were unknotted rings. This result excludes synapsis by way of a random collision of recombination sites, because the resulting entrapped supercoils would be converted into knots by recombination. Instead, the recombination sites must come together in the synaptic complex in an ordered fashion with a fixed number of supercoils between the sites. The linking number of the substrate DNA increases by four during recombination. Thus, in three successive rounds of inversion, the change in linking number was +4, +8, and +12, respectively. These results lead to a quantitative model for the mechanism of Gin recombination that includes the distribution of supercoils in the synaptic complex, their alteration by strand exchange, and specific roles for the two proteins needed for recombination.     

ATP-independent type II topoisomerase from trypanosomes.

We have characterized in Trypanosoma cruzi a DNA topoisomerase capable of decatenating complex trypanosomal kinetoplast DNA networks in the absence of ATP. The enzymatic activity requires Mg2+ and K+. Using a defined DNA topoisomer we showed that the linking number changes by steps of 2, which characterizes the enzyme as a type II topoisomerase. The enzyme can catenate supercoiled DNA molecules, unknot DNA, and cleave double-stranded DNA. The enzyme has no ATPase activity. The native enzyme has an Mr of about 200,000. Crude extracts and partially purified fractions contain an aggregating factor that can substitute spermidine in catenating reactions. Because of the presence of this factor, the kinetoplast DNA can only be decatenated by purified fractions. The enzyme is inhibited by certain drugs and provides a potential target for chemotherapy. Such an enzyme was also characterized in Trypanosoma equiperdum.     

Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments.

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.