Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ruv operon is induced by treatments that damage DNA and is regulated by the LexA repressor. It encodes two proteins, RuvA and RuvB, that are involved in DNA repair, recombination in RecE and RecF pathways, and mutagenesis. RuvB protein was previously purified and has ATP-binding activity and weak ATPase activity. To study the biochemical properties of RuvA and its interaction with RuvB, we purified RuvA protein to near homogeneity from an over-producing strain. RuvA bound more efficiently to single-stranded DNA than to double-stranded DNA. RuvA bound to DNA greatly enhanced the ATPase activity of RuvB; the enhancing effect of various forms of DNA was in the order of supercoiled DNA greater than single-stranded DNA greater than linear double-stranded DNA. UV irradiation further enhanced the ATPase stimulatory effect of supercoiled DNA dose dependently. The RuvA-RuvB complex has an activity that renatures the cruciform structure in supercoiled DNA. From these experiments and previous work, we infer that the RuvA-RuvB complex may promote branch migration in recombination and may correct irregular structures in DNA, such as cruciforms and hairpins, to facilitate DNA repair using ATP as the energy source.
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PMID:SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. 183 59

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.
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PMID:Purification of a RecA protein analogue from Bacillus subtilis. 315 34

Escherichia coli RecA protein plays a central role both in DNA repair and in recombination. We report biochemical properties of three new RecA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site. RecA694 had a wild-type phenotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange reaction. In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-stranded DNA (with and without single-stranded binding protein, SSB), double-stranded DNA, and ATP. DNA strand exchange defects were correlated with the inability of mutant proteins to displace SSB from DNA. For the recA659 mutation this inhibition was reversed by equimolar wild-type protein. In contrast, mixtures of either wild-type/RecA659 or wild-type/RecA611 proteins remained deficient in LexA cleavage, suggesting that the dominant negative phenotype of the mutant proteins may be a consequence of the formation heterologous RecA complexes. Various mutations in the putative DNA binding site of RecA protein altered ATP binding, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions. These results are consistent with the hypothesis that DNA binding to this site of RecA relays allosteric effects to several functional domains throughout the protein.
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PMID:Purification and biochemical characterization of Escherichia coli RecA proteins mutated in the putative DNA binding site. 813 49

The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.
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PMID:Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex. 982 66

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.
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PMID:Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo. 1242 42

The bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament. We examine RecA-DNA-ATP filaments complexed with LexA and RecX to shed more light on the active RecA filament. The LexA repressor and RecX, an inhibitor of RecA, both bind within the deep helical groove of the RecA filament. Residues on RecA that interact with LexA cannot be explained by the crystal filament, but can be properly positioned in an existing model for the active filament. We show that the strand exchange activity of RecA, which can be inhibited when RecX is present at very low stoichiometry, is due to RecX forming a block across the deep helical groove of the RecA filament, where strand exchange occurs. It has previously been shown that changes in the nucleotide bound to RecA are associated with large motions of RecA's C-terminal domain. Since RecX binds from the C-terminal domain of one subunit to the nucleotide-binding core of another subunit, a stabilization of RecA's C-terminal domain by RecX can likely explain the inhibition of RecA's ATPase activity by RecX.
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PMID:Complexes of RecA with LexA and RecX differentiate between active and inactive RecA nucleoprotein filaments. 1452 21

RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study of recA mutation in other organisms. 'Missing' recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi's RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the 'missing' phenotypes.
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PMID:Biochemical characterization of Borrelia burgdorferi's RecA protein. 2908 68

Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium smegmatis RecA foci localize to the cell poles during normal growth, and these structures disassemble and reassemble into several foci along the cell after the induction of DNA damage. Taken together, these data support the notion that the interaction of RecA with cardiolipin and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and may regulate recombinational DNA repair and the SOS response.
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PMID:The Anionic Phospholipids in the Plasma Membrane Play an Important Role in Regulating the Biochemical Properties and Biological Functions of RecA Proteins. 3072 69