POL32 , a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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The product of Saccharomyces cerevisiae WHIP/MGS1, a gene related to replication factor C genes, interacts functionally with DNA polymerase δ

Molecular Genetics and Genomics ◽

10.1007/s00438-002-0757-3 ◽

2002 ◽

Vol 268 (3) ◽

pp. 371-386 ◽

Cited By ~ 42

Author(s):

D. Branzei ◽

M. Seki ◽

F. Onoda ◽

T. Enomoto

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Replication Factor C ◽

Replication Factor ◽

Dna Polymerase Δ ◽

Factor C

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The TREX2 3′→ 5′ Exonuclease Physically Interacts with DNA Polymerase δ and Increases Its Accuracy

The Scientific World JOURNAL ◽

10.1100/tsw.2002.99 ◽

2002 ◽

Vol 2 ◽

pp. 275-281 ◽

Cited By ~ 15

Author(s):

Igor V. Shevelev ◽

Kristijan Ramadan ◽

Ulrich Hubscher

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Error Rates ◽

High Fidelity ◽

Replication Machinery ◽

Dna Polymerase Δ ◽

Pol I ◽

Calf Thymus ◽

Mutation Assay ◽

Forward Mutation

Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δdfrom isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δdhad a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δdwas fivefold more accurate than pol δiunder error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5724-5735.1992 ◽

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.

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Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.155-163.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 155-163 ◽

Cited By ~ 1

Author(s):

K Fien ◽

B Stillman

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Polymerase ◽

Replication Factor C ◽

Dna Polymerase Delta ◽

Replication Complex ◽

Leading Strand ◽

Factor C ◽

Sv40 Dna

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

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