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)

DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.
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PMID:DNA substrate specificity of DNA helicase E from calf thymus. 133 62

A novel DNA helicase has been isolated from Saccharomyces cerevisiae. This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC. Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl. The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP. It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM. The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP. The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.
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PMID:A Saccharomyces cerevisiae DNA helicase associated with replication factor C. 146 28

Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).
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PMID:Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. 167 Oct 45

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.
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PMID:Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. 196 33

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
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PMID:Further characterization of the human cell multiprotein DNA replication complex. 853 May 40

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

Ku is a heterodimeric protein composed of approximately 70- and approximately 80-kDa subunits (Ku70 and Ku80) originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. Ku has high binding affinity for DNA ends and that is why originally it was known as a DNA end binding protein, but now it is known to also bind the DNA structure at nicks, gaps, hairpins, as well as the ends of telomeres. It has been reported also to bind with sequence specificity to DNA and with weak affinity to RNA. Ku is an abundant nuclear protein and is present in vertebrates, insects, yeast, and worms. Ku contains ssDNA-dependent ATPase and ATP-dependent DNA helicase activities. It is the regulatory subunit of the DNA-dependent protein kinase that phosphorylates many proteins, including SV-40 large T antigen, p53, RNA-polymerase II, RP-A, topoisomerases, hsp90, and many transcription factors such as c-Jun, c-Fos, oct-1, sp-1, c-Myc, TFIID, and many more. It seems to be a multifunctional protein that has been implicated to be involved directly or indirectly in many important cellular metabolic processes such as DNA double-strand break repair, V(D)J recombination of immunoglobulins and T-cell receptor genes, immunoglobulin isotype switching, DNA replication, transcription regulation, regulation of heat shock-induced responses, regulation of the precise structure of telomeric termini, and it also plays a novel role in G2 and M phases of the cell cycle. The mechanism underlying the regulation of all the diverse functions of Ku is still obscure.
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PMID:Ku autoantigen: a multifunctional DNA-binding protein. 1075 64