Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex.

Plevani, Paolo; Foiani, Marco; Valsasnini, P.; Badaracco, Gianfranco; Cheriathundam, Elizabeth; Chang, Lucy M.S.

doi:10.1016/s0021-9258(18)88893-7

Cited by 111 publications

(20 citation statements)

References 27 publications

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“…T4 polynucleotide kinase was obtained from New England Biolabs (Beverly, MA). Yeast polymerase R-primase complex used in this study was purified as described (Plevani et al, 1985). The anti-pol R monoclonal antibody (MAb)-agarose column, used in the pol R-primase purification, was received as a gift from Dr. Lucy M. S. Chang of Uniformed Services Health Sciences University.…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Zhu

Biswas

1997

Biochemistry

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We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

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Section: Methodsmentioning

confidence: 99%

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Zhu

Biswas

1997

Biochemistry

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“…The predicted molecular mass of the POL12 gene product is 78.9 kDa, while the purified protein migrates as an 86-kDa polypeptide when analyzed by SDS-PAGE (7). Since most of our immunopurified Pol a-primase complex preparations contain a major 74-kDa protein band (53,62) and a panel of monoclonal antibodies was produced against this complex, it was of interest to verify whether some of these antibodies recognized the 86-kDa polypeptide in crude extracts when proteolysis was prevented.…”

Section: Resultsmentioning

confidence: 94%

“…The protein complex of the yeast Saccharomyces cerevisiae contains four polypeptides with apparent molecular masses of 180, 86, 58, and 48 kDa (7, 53). The p180 polypeptide has been shown to be the catalytic Pol a subunit, while the DNA primase is a heterodimer of the 58 and 48 kDa polypeptides (6,7,52,53). We have shown that the p48 subunit is sufficient for RNA primer synthesis in vitro, although an auxiliary role for the 58-kDa polypeptide in DNA primase activity in vivo cannot be excluded (62).…”

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confidence: 81%

The B Subunit of the DNA Polymerase α-Primase Complex in Saccharomyces cerevisiae Executes an Essential Function at the Initial Stage of DNA Replication

Foiani¹,

Marini²,

Gamba

et al. 1994

Molecular and Cellular Biology

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The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

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“…Specifically, most reports have demonstrated a group of large molecular weight polypeptides, ca. 125-180 kDa (1)(2)(3)(4)(5)(6)(7)(8)(9)(10), which contain the catalytic activity of polymerase a; two lower molecular weight polypeptides, ca. 55 and 46 kDa (1-10), which contain DNA primase activity (1,3,11); and the presence of an intermediate molecular weight polypeptide, ca.…”

Section: Introductionmentioning

confidence: 99%

“…125-180 kDa (1)(2)(3)(4)(5)(6)(7)(8)(9)(10), which contain the catalytic activity of polymerase a; two lower molecular weight polypeptides, ca. 55 and 46 kDa (1-10), which contain DNA primase activity (1,3,11); and the presence of an intermediate molecular weight polypeptide, ca. 70 kDa (1-3,6-8,10), which exhibits no known enzymological activity and has been shown in various systems to be a tightly associated component of the DNA polymerase/DNA primase complex (6,7,10,12).…”

Section: Introductionmentioning

confidence: 99%

The evolutionary conservation of DNA polymerase α

1988

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The evolutionary conservation of DNA polymerase alpha was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase alpha monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. The most highly cross-reactive antibody recognizes a unique epitope on a 165-180 kDa catalytic polypeptide in cell lysates from several eukaryotic sources, as distant from man as the amphibians. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase alpha catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA polymerase alpha among vertebrates and the conservation of specific genetic sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

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Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex.

Cited by 111 publications

References 27 publications

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

The B Subunit of the DNA Polymerase α-Primase Complex in Saccharomyces cerevisiae Executes an Essential Function at the Initial Stage of DNA Replication

The evolutionary conservation of DNA polymerase α

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