Purification and Characterization of Rad3 ATPase/DNA helicase from Saccharomyces cerevisiae

Harosh, Itzik; Naumovski, Louie; Friedberg, Errol C.

doi:10.1016/s0021-9258(19)47095-6

Cited by 56 publications

(10 citation statements)

References 21 publications

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“…The association of an ATPase with the multiprotein yeast DNA polymerase a, with an RP-A-dependent duplex unwinding activity, could have important implications in the function of this multiprotein complex in chromosomal DNA replication. Three helicases have recently been described (Suginoetal., 1986;Sung etal., 1987a, b;Abussekhara, 1989;Harosh et al, 1989). Two independent helicases from mammalian sources have been described; however, the structure and function of these helicases are still not complete (Seo et al, 1991; Hubscher & Stalder, 1986).…”

Section: Discussionmentioning

confidence: 99%

“…1 Abbreviations: pol a, DNA polymerase a; pol 6, DNA polymerase (5; pol t, DNA polymerase e; Tris, tris(hydroxymethyl)aminomethane; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; ATP, adenosine 5'-triphosphate; ATPase, adenosinetriphosphatase; ss-DNA, single-stranded DNA; dsDNA, double-stranded DNA; RF-C, replication factor C; RP-A, replication protein A. 0006-2960/93/0432-3020S04.00/0 (Sung et al, 1987a,b;Harosh et al, 1989). Sugino et al (1986) have reported the purification of a 63-kDa helicase.…”

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confidence: 99%

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Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

Biswas

Ewing²,

Biswas³

1993

Biochemistry

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We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

Biswas

Ewing²,

Biswas³

1993

Biochemistry

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“…Compound CC-1065 was generously provided by Dr. J. Patrick McGovren, The Upjohn Co., Kalamazoo, MI. Homogeneous preparations of Rad3 protein were obtained as previously described (Harosh et al, 1989;Naegeli et al, 1992a).…”

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confidence: 99%

“…A 32P-labeled 206-mer oligonucleotide was produced by single-stranded polymerase chain reaction and annealed to M13mpl8 single-stranded DNA as described (Naegeli et al, 1992a). The 30-mer DNA primer (Harosh et al, 1989) was 5' end-labeled by T4 polynucleotide kinase prior to being annealed to M13mpl8 single-stranded DNA (Naegeli et al, 1992a). (dA)25-3o and (dT)25-3o were dephosphorylated and 5' end-labeled as previously described (Naegeli et al, 1992b).…”

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confidence: 99%

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Inhibition of Rad3 DNA helicase activity by DNA adducts and abasic sites: implications for the role of a DNA helicase in damage-specific incision of DNA

Naegeli¹,

Bardwell²,

Friedberg³

1993

Biochemistry

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The yeast nucleotide excision repair gene RAD3 is absolutely required for damage-specific incision of DNA. Rad3 protein is a DNA helicase, and previous studies have shown that its catalytic activity is inhibited by ultraviolet (UV) radiation damage. This inhibition is observed when base damage is confined to the DNA strand on which Rad3 protein binds and translocates, and not when damage is present exclusively on the complementary strand. In the present study, we show that Rad3 DNA helicase activity is inhibited in an identical strand-specific fashion by bulky base adducts formed by treating DNA with the antineoplastic agent cisplatin or the antibiotic compound CC-1065, which alter the secondary structure of DNA in different ways. In addition, Rad3 helicase activity is inhibited by small adducts generated by treatment of DNA with diethyl sulfate and by the presence of sites at which pyrimidines have been lost (abasic sites). No inhibition of Rad3 helicase activity was detected when DNA was methylated at various base positions. Cisplatin-modified single-stranded DNA and poly(deoxyuridylic acid) containing abasic sites are more effective competitors for Rad3 helicase activity than their undamaged counterparts, suggesting that Rad3 protein is sequestered at such lesions, resulting in the formation of stable Rad3 protein-DNA complexes. The observations of strand-specific inhibition of Rad3 helicase activity and the formation of stable complexes with the covalently modified strand suggest a general mechanism by which the RAD3 gene product may be involved in nucleotide excision repair in yeast.

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Eukaryotic DNA repair: Glimpses through the yeast Saccharomyces cerevisiae

Friedberg

1991

BioEssays

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Purification and Characterization of Rad3 ATPase/DNA helicase from Saccharomyces cerevisiae

Cited by 56 publications

References 21 publications

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

Inhibition of Rad3 DNA helicase activity by DNA adducts and abasic sites: implications for the role of a DNA helicase in damage-specific incision of DNA

Eukaryotic DNA repair: Glimpses through the yeast Saccharomyces cerevisiae

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