scholarly journals The role of DNA polymerase alpha in the control of mutagenesis in Saccharomyces cerevisiae cells starved for nutrients

2011 ◽  
Vol 9 (1) ◽  
pp. 53-61
Author(s):  
Nora Babudri ◽  
Alessandro Achilli ◽  
Chiara Martinelli ◽  
Elizabeth Moore ◽  
Hovirag Lancioni ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Yeast Cells ◽  
Dna Polymerase Alpha ◽  
Dna Polymerase Α ◽  
Dividing Cells ◽  
Base Selection

In nature, microorganisms experience numerous environmental stresses and generally grow poorly most of the time. In the last two decades it has become evident that mutations arise not only in actively dividing cells but also in nonreplicating or slowly replicating cells starved for nutrients. In yeast, precise base selection and proofreading by replicative DNA polymerases δ and ε keep starvation-associated mutagenesis (SAM) at basal levels. Less is known about the role of replicative DNA polymerase α (Pol α). Here we provide evidence that Pol α is involved in the control of SAM in yeast cells starved for adenine by participation in sporadic replication and/or DNA repair under these conditions. 

scholarly journals Role of Translesion Synthesis DNA Polymerases in DNA Replication in the Presence of a Weak DNA Polymerase δ in Saccharomyces cerevisiae

2018 ◽  
Vol 8 (2) ◽  
pp. 754-754
Author(s):  
Likui Zhang ◽  
Yanchao Huang ◽  
Xinyuan Zhu ◽  
Yuxiao Wang ◽  
Haoqiang Shi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Dna Polymerase Δ

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo

1992 ◽  
Vol 12 (12) ◽  
pp. 5724-5735
Author(s):  
J Miles ◽  
T Formosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Yeast Cells ◽  
Chromosome Loss ◽  
Dna Metabolism ◽  
Checkpoint Gene ◽  
Dna Polymerase Alpha ◽  
In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

scholarly journals Role of Translesion Synthesis DNA Polymerases in DNA Replication in the Presence of a Weak DNA Polymerase δ in Saccharomyces cerevisiae

2017 ◽  
Vol 8 (2) ◽  
pp. 754-754
Author(s):  
Likui Zhang ◽  
Yanchao Huang ◽  
Xinyuan Zhu ◽  
Yuxiao Wang ◽  
Haoqiang Shi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Dna Polymerase Δ

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 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 Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo.

1992 ◽  
Vol 12 (12) ◽  
pp. 5724-5735 ◽  
Author(s):  
J Miles ◽  
T Formosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Yeast Cells ◽  
Chromosome Loss ◽  
Dna Metabolism ◽  
Checkpoint Gene ◽  
Dna Polymerase Alpha ◽  
In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. L1174-L1180 ◽  
Author(s):  
M. Takeoka ◽  
W. F. Ward ◽  
H. Pollack ◽  
D. W. Kamp ◽  
R. J. Panos
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Epithelial Cells ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Alveolar Epithelial ◽  
Radiation Induced ◽  
Dna Strand

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

scholarly journals The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 648
Author(s):  
del Prado ◽  
Santos ◽  
Lázaro ◽  
Salas ◽  
de Vega
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Structural Changes ◽  
Binding Capacity ◽  
Dna Polymerases ◽  
Crystallographic Structure ◽  
Genome Replication ◽  
Terminal Protein ◽  
Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

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