Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.
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PMID:Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. 798 72

Saccharomyces cerevisiae RAD51 gene is required for genetic recombination and recombinational repair of DNA strand breaks. Rad51 protein has a DNA-dependent ATPase activity, and it catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules. We show here that the rad51 Arg-191 protein, which is devoid of ATPase activity, mediates the pairing and strand exchange reaction upon binding ATP. In addition, the wild type Rad51 protein can catalyze pairing and strand exchange in the presence of the nonhydrolyzable ATP analogues adenylyl-imidodiphosphate and adenosine 5'-O-thiotriphosphate. Thus, homologous pairing and the unidirectional transfer of greater than 5 kilobases of DNA can occur efficiently without the need for nucleotide hydrolysis. Consistent with the results from the biochemical analyses, expression of the rad51 Arg-191 protein in a rad51 null mutant confers normal cellular resistance to the DNA damaging agent methylmethane sulfonate, suggesting that nucleotide binding by Rad51 is sufficient for biological function.
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PMID:Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis. 891 Apr 3

The human testis Rad51 protein, a structural homolog of E. coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using circular ssDNA and linear dsDNA (3.0 kb in length), we demonstrate that hRad51 promotes homologous pairing and strand exchange reactions in vitro. Joint molecule formation was dependent upon ATP hydrolysis and DNA homology and was stimulated by the single-strand DNA-binding protein RP-A. In these reactions, the 5' terminus of the complementary strand of the linear duplex was efficiently transferred to the ssDNA. However, under standard conditions, extensive strand exchange was not observed. These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer. It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins.
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PMID:Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. 892 43

Protein-promoted DNA strand exchange requires formation of an active presynaptic complex between the DNA-pairing protein and single-stranded DNA (ssDNA). Formation of such a contiguous filament is stimulated by a ssDNA-binding protein. Here, the effects of replication protein A (RPA) on presynaptic complex formation and DNA strand exchange activities of Rad51 protein were examined. Presynaptic complex formation was assessed by measuring ATP hydrolysis. With phiX174 ssDNA, the ATPase activity of Rad51 protein is stimulated approximately 1.4-fold by RPA, provided that Rad51 protein is in excess of the ssDNA concentration; otherwise, RPA inhibits ATPase activity. In contrast, with ssDNA devoid of secondary structure (poly(dT), poly(dA), poly(dI), and etheno-M13 DNA), RPA does not stimulate the already elevated ATPase activity of Rad51 protein, but inhibits activity at low Rad51 protein concentrations. These results suggest that Rad51 protein and RPA exclude one another from ssDNA by competing for the same binding sites and that RPA exerts its effect on presynaptic complex formation by eliminating secondary structure to which Rad51 protein is bound nonproductively. DNA strand exchange catalyzed by Rad51 protein is also greatly stimulated by RPA. The optimal stoichiometry for stimulation is approximately 20-30 nucleotides of ssDNA/RPA heterotrimer. The ssDNA-binding protein of Escherichia coli can substitute for RPA, showing that the role of RPA is not specific. We conclude that RPA affects both presynaptic complex formation and DNA strand exchange via changes in DNA structure, employing the same mechanism used by the ssDNA-binding protein to effect change in E. coli RecA protein activity.
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PMID:A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. 906 63

With the discovery that the Saccharomyces cerevisiae Rad51 protein is both structurally and functionally similar to the Escherichia coli RecA protein, the RecA paradigm for homologous recombination was extended to the Eucarya. The ubiquitous presence of RecA and Rad51 protein homologs raises the question of whether this archetypal protein exists within the third domain of life, the Archaea. Here we present the isolation of a Rad51/RecA protein homolog from the archaeon Sulfolobus solfataricus, and show that this protein, RadA, possesses the characteristics of a DNA strand exchange protein: The RadA protein is a DNA-dependent ATPase, forms a nucleoprotein filament on DNA, and catalyzes DNA pairing and strand exchange.
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PMID:RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange. 957 41

The Saccharomyces cerevisiae RAD51 and RAD54 genes are both required for the occurrence of homologous recombination and for the repair of double-stranded DNA breaks. Previous studies have indicated that Rad51 protein, together with the single-stranded DNA-binding factor replication protein A (RPA), can promote the formation of heteroduplex DNA, which is a key intermediate in homologous recombination. Here we report the purification of the Rad54 protein to near homogeneity and the biochemical testing of its molecular function. We find that Rad54 protein possesses a double-stranded DNA-dependent ATPase activity, and that it interacts with the Rad51 protein. Addition of Rad54 protein to reactions containing Rad51 strongly stimulates the rate of pairing between homologous single-stranded and double-stranded DNA molecules. We conclude that Rad54 acts to overcome kinetic impediments that would limit homologous DNA pairing between recombining chromosomes in vivo.
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PMID:Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. 959 Jun 97

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.
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PMID:Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila. 962 14

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.
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PMID:A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon. 1006 83

Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA.
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PMID:Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. 1050 8

The Rad51 protein in eukaryotic cells is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Several proteins showing sequence similarity to Rad51 have previously been identified in both yeast and human cells. In Saccharomyces cerevisiae, two of these proteins, Rad55p and Rad57p, form a heterodimer that can stimulate Rad51-mediated DNA strand exchange. Here, we report the purification of one of the representatives of the RAD51 family in human cells. We demonstrate that the purified RAD51L3 protein possesses single-stranded DNA binding activity and DNA-stimulated ATPase activity, consistent with the presence of "Walker box" motifs in the deduced RAD51L3 sequence. We have identified a protein complex in human cells containing RAD51L3 and a second RAD51 family member, XRCC2. By using purified proteins, we demonstrate that the interaction between RAD51L3 and XRCC2 is direct. Given the requirements for XRCC2 in genetic recombination and protection against DNA-damaging agents, we suggest that the complex of RAD51L3 and XRCC2 is likely to be important for these functions in human cells.
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PMID:The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2. 1087 7


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