scholarly journals The 3′→5′ Exonucleases of DNA Polymerases δ and ɛ and the 5′→3′ Exonuclease Exo1 Have Major Roles in Postreplication Mutation Avoidance in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (3) ◽  
pp. 2000-2007 ◽  
Author(s):  
Hiep T. Tran ◽  
Dmitry A. Gordenin ◽  
Michael A. Resnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Mutation Rate ◽  
Mutation Detection ◽  
Dna Polymerases ◽  
Genetic Interactions ◽  
Detection Systems ◽  
Mutation Avoidance ◽  
Highly Sensitive

ABSTRACT Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5′→3′ Exo1 and the 3′→5′ DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase ɛ (pol2-4) and δ (pol3-01) mutants defective in 3′→5′ proofreading exonuclease, mutants defective in the 5′→3′ exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.

scholarly journals DNA polymerase δ proofreads errors made by DNA polymerase ε

2020 ◽  
Vol 117 (11) ◽  
pp. 6035-6041 ◽  
Author(s):  
Chelsea R. Bulock ◽  
Xuanxuan Xing ◽  
Polina V. Shcherbakova
Keyword(s):  
Dna Polymerase ◽  
Mutation Rate ◽  
Direct Evidence ◽  
Dna Polymerases ◽  
Dna Polymerase Δ ◽  
Mutation Avoidance ◽  
The One

During eukaryotic replication, DNA polymerases ε (Polε) and δ (Polδ) synthesize the leading and lagging strands, respectively. In a long-known contradiction to this model, defects in the fidelity of Polε have a much weaker impact on mutagenesis than analogous Polδ defects. It has been previously proposed that Polδ contributes more to mutation avoidance because it proofreads mismatches created by Polε in addition to its own errors. However, direct evidence for this model was missing. We show that, in yeast, the mutation rate increases synergistically when a Polε nucleotide selectivity defect is combined with a Polδ proofreading defect, demonstrating extrinsic proofreading of Polε errors by Polδ. In contrast, combining Polδ nucleotide selectivity and Polε proofreading defects produces no synergy, indicating that Polε cannot correct errors made by Polδ. We further show that Polδ can remove errors made by exonuclease-deficient Polε in vitro. These findings illustrate the complexity of the one-strand–one-polymerase model where synthesis appears to be largely divided, but Polδ proofreading operates on both strands.

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

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

Spontaneous Frameshift Mutations in Saccharomyces cerevisiae: Accumulation During DNA Replication and Removal by Proofreading and Mismatch Repair Activities

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Christopher N Greene ◽  
Sue Jinks-Robertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Wild Type Strain ◽  
Dna Polymerases ◽  
Wild Type ◽  
Triple Mutant ◽  
Repair Capacity ◽  
Frameshift Mutations ◽  
Dna Polymerization ◽  
In The Wild

Abstract The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery. To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2ΔBgl +1 frameshift allele. Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases δ and ε (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed. Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity. Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair. Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast.

A method to select for mutator DNA polymerase δs in Saccharomyces cerevisiae

Genome ◽  
2006 ◽  
Vol 49 (4) ◽  
pp. 403-410 ◽  
Author(s):  
Kelly Murphy ◽  
Hariyanto Darmawan ◽  
Amy Schultz ◽  
Elizabeth Fidalgo da Silva ◽  
Linda J Reha-Krantz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Structural Information ◽  
Bacteriophage T4 ◽  
Dna Polymerases ◽  
Genetic Selection ◽  
Selection Procedure ◽  
Dna Polymerase Δ ◽  
Peptide Motifs ◽  
New Yeast

Proofreading DNA polymerases share common short peptide motifs that bind Mg2+ in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase δ mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase δ mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.Key words: DNA polymerase proofreading, genetic selection for mutator mutants, fidelity of DNA replication, yeast.

Sequence Composition and Context Effects on the Generation and Repair of Frameshift Intermediates in Mononucleotide Runs in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 571-578
Author(s):  
Brian D Harfe ◽  
Sue Jinks-Robertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Context Effects ◽  
Complete Elimination ◽  
Sequence Context ◽  
Frameshift Mutations ◽  
Sequence Composition ◽  
Overall Efficiency ◽  
Polymerase Slippage

Abstract DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys+ revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.

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 Study of dogfish (Scyliorhinus caniculus) deoxyribonucleic acid polymerase α and β. Extraction, separation, characterization and changes during spermatogenesis

1980 ◽  
Vol 189 (3) ◽  
pp. 635-639 ◽  
Author(s):  
M Philippe ◽  
P Chevaillier
Keyword(s):  
Dna Polymerase ◽  
Sucrose Gradient ◽  
Deoxyribonucleic Acid ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Polymerase Gamma ◽  
Polymerase Beta ◽  
Dna Polymerase Gamma ◽  
Highly Sensitive ◽  
Extraction Separation

DNA polymerase activity was extracted from testis cells of the dogfish Scyliorhinus caniculus. On a sucrose gradient, two main peaks could be separated, corresponding to DNA polymerases beta (3.8 S) and alpha (7.5 S). DNA polymerase gamma could also be detected when poly(A) . (dT)12 was used as template. The properties of alpha and beta polymerases of this primitive vertebrate were similar to those generally described, especially in mammals. The beta enzyme was highly sensitive to N-ethylmaleimide, however, and could use poly(dT) . poly(A) as template. Polymerase alpha was present in spermatogonia, spermatocytes and spermatids. Activity was maximal in spermatocytes. DNA polymerase beta was present in all testis cells with similar activities in spermatogonia and spermatocytes. Decreased activities were observed during spermiogenesis. Some activity remained associated with the chromatin fraction of mature sperm cells.

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