scholarly journals Fidelity studies of the human DNA polymerase alpha. The most conserved region among alpha-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis

1993 ◽  
Vol 268 (15) ◽  
pp. 11041-11049
Author(s):  
W.C. Copeland ◽  
N.K. Lam ◽  
T.S. Wang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase Alpha ◽  
Human Dna ◽  
Conserved Region

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

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 256-264 ◽  
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.

scholarly journals Involvement of the essential yeast DNA polymerases in induced gene conversion.

1999 ◽  
Vol 46 (4) ◽  
pp. 862-872 ◽  
Author(s):  
A Hałas ◽  
A Ciesielski ◽  
J Zuk
Keyword(s):  
Dna Synthesis ◽  
Gene Conversion ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Polymerase Alpha ◽  
Genes Encoding ◽  
Mitotic Gene Conversion ◽  
Dna Strand

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.

scholarly journals DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (1) ◽  
pp. 496-505 ◽  
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nonpermissive Temperature ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

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

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 256-264
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

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.

scholarly journals DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 496-505
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nonpermissive Temperature ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

Sign in / Sign up

Export Citation Format

Share Document