Catalytic subunit of human DNA polymerase alpha overproduced from baculovirus-infected insect cells. Structural and enzymological characterization.

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

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Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.94 ◽

1996 ◽

Vol 16 (1) ◽

pp. 94-104 ◽

Cited By ~ 27

Author(s):

F Stadlbauer ◽

C Voitenleitner ◽

A Brückner ◽

E Fanning ◽

H P Nasheuer

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Polymerase Alpha ◽

Cell Extracts ◽

Human Dna ◽

Sv40 Dna ◽

Hybrid Complexes

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.

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Structure of the Gene for the Catalytic Subunit of Human DNA Polymerase δ (POLD1)

Genomics ◽

10.1006/geno.1995.1169 ◽

1995 ◽

Vol 28 (3) ◽

pp. 411-419 ◽

Cited By ~ 18

Author(s):

Long-Sheng Chang ◽

Lingyun Zhao ◽

Lingyun Zhu ◽

Meei-Ling Chen ◽

Marietta Y.W.T. Lee

Keyword(s):

Dna Polymerase ◽

Catalytic Subunit ◽

Dna Polymerase Δ ◽

Human Dna

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Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000-dalton catalytic subunit of DNA polymerase alpha.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.83.20.7608 ◽

1986 ◽

Vol 83 (20) ◽

pp. 7608-7612 ◽

Cited By ~ 17

Author(s):

G. Seal ◽

M. A. Sirover

Keyword(s):

Dna Polymerase ◽

Base Excision Repair ◽

Excision Repair ◽

Catalytic Subunit ◽

Dna Glycosylase ◽

Repair Enzyme ◽

Uracil Dna Glycosylase ◽

Dna Polymerase Alpha ◽

Physical Association ◽

Base Excision

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Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80506-7 ◽

1993 ◽

Vol 268 (32) ◽

pp. 24163-24174

Author(s):

Q Dong ◽

W.C. Copeland ◽

T.S. Wang

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Dna Polymerase Alpha ◽

Human Dna

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Chlorodeoxyadenosine and arabinosylcytosine in patients with acute myelogenous leukemia: pharmacokinetic, pharmacodynamic, and molecular interactions

Blood ◽

10.1182/blood.v87.1.256.256 ◽

1996 ◽

Vol 87 (1) ◽

pp. 256-264 ◽

Cited By ~ 104

Author(s):

V Gandhi ◽

E Estey ◽

MJ Keating ◽

A Chucrallah ◽

W Plunkett

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Ribonucleotide Reductase ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Biochemical Modulation ◽

Dna Polymerase Alpha ◽

Human Dna ◽

Acute Myelogenous ◽

Dna Primers

Abstract The effectiveness of arabinosylcytosine (ara-C) for the treatment of acute myelogenous leukemia (AML) depends on the formation of its active metabolite, the triphosphate of ara-C (ara-CTP). Using biochemical modulation strategies to increase the accumulation of ara-CTP in leukemia blasts, a clinical protocol was designed combining 2- chlorodeoxyadenosine (CdA), an inhibitor of ribonucleotide reductase, and ara-C for adults with AML. The protocol stipulated an infusion of 1 g/m2 of ara-C over 2 hours on day 1. A continuous infusion of CdA (12 mg/m2/d) begun 24 hours later and continued for 5 days. Identical doses of ara-C were administered on days 3, 4, 5, and 6. Pharmacokinetic and pharmacodynamic interactions between CdA and ara-C during therapy were investigated. To complement these studies, molecular actions of the triphosphate of ara-C and CdA on DNA extension by human DNA polymerase alpha in an in vitro model system was conducted. In the circulating leukemia blasts of 7 of the 9 patients studied, ara-CTP pharmacokinetics showed a median 40% increase in the rate of ara-CTP accumulation after 24 hours of CdA infusion. The ex vivo effect of CdA on accumulation of ara-CTP in AML blasts was similar to that during therapy except that the enhancement was less. The DNA synthetic capacity of the circulating blasts was inhibited to a greater extent by administration of CdA and ara-C in combination than by either one alone. Additionally the lowered level of DNA synthesis was maintained until the next infusion of ara-C. Endogenous levels of deoxynucleotides increased 24 hours after ara-C infusion. Administration of CdA in general lowered the concentrations of all dNTPs. DNA pol alpha incorporated CdATP and ara-CTP with high affinity in a DNA primer extending over an oligonucleotide template of defined sequence. Human DNA polymerase alpha extended DNA primers terminated by CdA monophosphate (CdAMP) at its 3′-end by incorporating ara-C monophosphate (ara-CMP). The tandem incorporation of CdAMP and ara-CMP resulted in nearly complete inhibition of DNA primer extension. The insertion of two analogs in sequence, inhibition of ribonucleotide reductase, and the metabolic potentiation of ara-CTP by CdA infusion may be responsible for sustained inhibition of DNA synthesis in the circulating leukemia blasts during therapy with this combination regimen.

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