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)

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.
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PMID:Domain structure of phage P4 alpha protein deduced by mutational analysis. 763 18

A yeast gene has been identified by screening for DNA replication mutants using a permeabilized cell replication assay. The mutant is temperature sensitive for growth and shows a cell cycle phenotype typical of DNA replication mutants. RNA synthesis is normal in the mutant but DNA synthesis ceases upon shift to the nonpermissive temperature. The DNA2 gene was cloned by complementation of the dna2ts gene phenotype. The gene is essential for viability. The gene encodes a 172-kDa protein with characteristic DNA helicase motifs. A hemagglutinin epitope-Dna2 fusion protein was prepared and purified by conventional and immunoaffinity chromatography. The purified protein is a DNA-dependent ATPase and has 3' to 5' DNA helicase activity specific for forked substrates. A nuclease activity that endonucleolytically cleaves DNA molecules having a single-stranded 5' tail adjacent to a duplex region copurifies through all steps with the fusion protein.
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PMID:A yeast gene required for DNA replication encodes a protein with homology to DNA helicases. 764 70

The RAD25 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and, in addition, is essential for viability. RAD25 shares a high degree of homology with the human ERCC3/XPBC-encoded protein, and the yeast and human proteins resemble one another in containing the conserved ATPase/DNA helicase sequence motifs. To determine the nature of the essential role of RAD25, we have isolated a recessive temperature-sensitive conditional lethal mutation of the gene and have examined its effect on transcription. Upon shift to the nonpermissive temperature, the rad25 temperature-sensitive (ts) mutant stops growth rapidly and shows a large decrease in the synthesis of poly(A)+ RNA. Transcription of a large number of yeast genes, including HIS3, TRP3, STE2, MET19, RAD23, CDC9, and ACT1 is inhibited at the restrictive temperature in the rad25 ts mutant, and the galactose-inducible synthesis of GAL7 and GAL10 mRNAs is also severely affected by the loss of RAD25 activity. These findings implicate a general requirement of RAD25 in RNA polymerase II transcription.
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PMID:The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II. 769 49

We have analyzed the contributions of specific domains of DnaB helicase to its quaternary structure and multienzyme activities. Highly purified tryptic fragments containing various domains of DnaB helicase were prepared. Fragment I lacks 14 amino acid (aa) residues from the N-terminal of DnaB helicase. Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I. The single-stranded DNA-dependent ATPase and DNA helicase activities of DnaB helicase and its fragments were examined in detail. The ATPase activities of native DnaB helicase and fragment I were comparable; however, the ATPase activity of fragment II was somewhat diminished. Unlike the ATPase activity, the DNA helicase activity was totally abolished in fragment II and was not complemented by the addition of equimolar fragment III. Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzymatic activities. Fragment I had a hexameric structure similar to that observed with DnaB helicase in both size exclusion HPLC (SE-HPLC) and chemical cross-linking studies. SE-HPLC analysis indicated that fragment II had an apparent hexameric form. However, a detailed chemical cross-linking analysis showed that it formed stable dimers but the formation of a stable hexamer was severely impaired. Thus, the N-terminal domain appeared to have a strong influence on the hexamer formation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structure and function of Escherichia coli DnaB protein: role of the N-terminal domain in helicase activity. 772 81

We present a preliminary biochemical characterization of two simian virus 40 mutants that affect different T antigen replication functions. SV40 T antigen mutants dl1135 (delta 17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography. With each of the purified proteins, we examined some of the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays. Consistent with previous studies, we found that the 5080 protein is defective for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization. However, this mutant retains some sequence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one of several mutants with an altered amino-terminal domain which can replicate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affects a T antigen enzymatic function directly required for viral DNA synthesis, dl1135 may alter an activity required to prepare the cell for viral replication.
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PMID:T antigens encoded by replication-defective simian virus 40 mutants dl1135 and 5080. 779 27

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.
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PMID:Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B. 779 22

The T4 gene 59 protein (gp59) serves as an accessory protein to the essential T4-encoded DNA helicase, the gene 41 protein (gp41). gp59 stimulates gp41-dependent DNA synthesis reactions by promoting the assembly of gp41 onto single-stranded DNA (ssDNA), where the enzyme is activated to perform its DNA helicase functions. To better understand the mechanism of helicase-ssDNA assembly, we have studied the effects of gp59 on the intrinsic and ssDNA-stimulated ATPase activities of gp41. Our results indicate that gp59 exerts a direct effect upon the conformation and ATPase activity of gp41, by increasing the affinity of gp41 for ATP. In addition, we find that gp59 is nearly essential for promoting the assembly of gp41 onto ssDNA molecules that are covered with saturating amounts of the T4-encoded helix-destabilizing protein, gene 32 protein (gp32). Results of protein affinity chromatography experiments suggest that gp59 contains distinct binding sites for gp41 and gp32 and may therefore act as a molecular adapter between the helicase and helix-destabilizing proteins. Together, the data indicate that specific gp59-gp41 and gp59-gp32 protein-protein interactions both play important roles in the assembly of the helicase onto single-stranded DNA.
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PMID:The gene 59 protein of bacteriophage T4 modulates the intrinsic and single-stranded DNA-stimulated ATPase activities of gene 41 protein, the T4 replicative DNA helicase. 780 35

We have purified a DNA dependent ATPase/DNA helicase, DNA helicase B, from S. cerevisiae. Helicase B was a 129-kDa polypeptide. The ATPase activity of helicase B was strongly DNA dependent. The DNA helicase activity was stimulated by yeast replication protein A, indicating a probable function in DNA replication. Helicase B showed a 5'-->3' polarity of movement. Protein sequencing indicated that helicase B was identical to a hypothetical 127-kDa polypeptide encoded by yORF61, located 5' upstream of the BMH1 locus in chromosome V. The protein sequence contained a "type I ATP/GTP binding motif" and other helicase-like motifs and the expressed protein exhibited helicase activity. Thus, we concluded that yORF61 is the gene for helicase B and will be referred to as HCSB.
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PMID:Biochemical and genetic characterization of a replication protein A dependent DNA helicase from the yeast, Saccharomyces cerevisiae. 783 96

Genes 41 and 59 of bacteriophage T4 are involved in DNA recombination as well as in DNA replication. The 41 protein has a DNA helicase activity. The 59 protein has been recently purified and found to have a specific affinity for both 32 protein (single-stranded DNA-binding protein) and 41 protein (Yonesaki 1994, J. Biol. Chem. 269, 1284-1289). We examined the effects of 59 protein on ssDNA-dependent ATPase activity and DNA helicase activity of 41 protein in the presence or absence of 32 protein. The ATPase activity of 41 protein was strongly inhibited by 32 protein over a wide range of amounts from subsaturation to oversaturation of ssDNA. The 32 protein was also inhibitory toward DNA helicase activity. Addition of 59 protein effectively eliminated these inhibitory effects of 32 protein. Moreover, 59 protein facilitated 41 protein to overcome the barrier to initiate the unwinding reaction with a duplex flanking a single-stranded DNA gap. Intriguingly, 32 protein at an amount optimal for saturation of ssDNA stimulated the overcoming of the barrier when 59 protein was present. For the best circumvention of this initiation barrier, only eight monomers of 59 protein/one DNA substrate molecule containing 2900 nucleotides of ssDNA were required. These results strongly suggest that 59 protein modulates 41 protein activities by forming a complex with 41 protein and that 41 protein can produce recombinogenic ssDNA with the aid of 32 and 59 proteins.
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PMID:Functional interactions of gene 32, 41, and 59 proteins of bacteriophage T4. 785 26

A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biopsied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E1 ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E1 ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells.
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PMID:Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: a rapid multiplex polymerase chain reaction approach. 785 62


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