DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

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The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

Genetics ◽

10.1093/genetics/139.4.1483 ◽

1995 ◽

Vol 139 (4) ◽

pp. 1483-1494 ◽

Cited By ~ 2

Author(s):

Y Cao ◽

T Kogoma

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Dna Polymerase ◽

Reca Protein ◽

Minor Role ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Polymerase I ◽

Recombination Repair ◽

Polymerase I

Abstract The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

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DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.11.3772 ◽

1988 ◽

Vol 85 (11) ◽

pp. 3772-3776 ◽

Cited By ~ 69

Author(s):

A. Pizzagalli ◽

P. Valsasnini ◽

P. Plevani ◽

G. Lucchini

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Dna Polymerase ◽

Dna Polymerases ◽

Temperature Sensitive ◽

Protein Homology ◽

Dna Polymerase I ◽

Sequence Mapping ◽

Polymerase I ◽

I Gene

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Two Temperature-Sensitive polA Mutants: An Approach to the Role in Vivo of DNA Polymerase I

Molecular Mechanisms for Repair of DNA ◽

10.1007/978-1-4684-2895-7_27 ◽

1975 ◽

pp. 209-211

Author(s):

Dennis G. Uyemura ◽

E. B. Konrad ◽

I. R. Lehman

Keyword(s):

Dna Polymerase ◽

Temperature Sensitive ◽

Dna Polymerase I ◽

Polymerase I ◽

Two Temperature

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Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽

10.1093/genetics/124.2.213 ◽

1990 ◽

Vol 124 (2) ◽

pp. 213-220 ◽

Cited By ~ 2

Author(s):

L J Reha-Krantz

Keyword(s):

Dna Polymerase ◽

Bacteriophage T4 ◽

Rnase H ◽

Ribonuclease H ◽

Temperature Sensitive ◽

Polymerase Gene ◽

Dna Polymerase I ◽

E Coli ◽

Intragenic Complementation ◽

Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

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Crystallization and preliminary X-ray analysis of thePlasmodium falciparumapicoplast DNA polymerase

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15002423 ◽

2015 ◽

Vol 71 (3) ◽

pp. 333-337 ◽

Cited By ~ 2

Author(s):

Morgan E. Milton ◽

Jun-yong Choe ◽

Richard B. Honzatko ◽

Scott W. Nelson

Keyword(s):

Dna Polymerase ◽

Structural Information ◽

Klenow Fragment ◽

X Ray Diffraction ◽

Dna Polymerase I ◽

X Ray ◽

Cell Parameters ◽

A Genome ◽

Reported Data ◽

Polymerase I

Infection by the parasitePlasmodium falciparumis the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment ofEscherichia coliDNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals ofP. falciparumapPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space groupP6522 (unit-cell parametersa=b= 141.8,c= 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress.

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New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

MGG Molecular & General Genetics ◽

10.1007/bf00384381 ◽

1982 ◽

Vol 187 (1) ◽

pp. 42-46 ◽

Cited By ~ 35

Author(s):

Lawrence B. Dumas ◽

Joan P. Lussky ◽

Elizabeth J. McFarland ◽

Janis Shampay

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants

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Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽

10.1093/genetics/148.3.989 ◽

1998 ◽

Vol 148 (3) ◽

pp. 989-1005 ◽

Cited By ~ 5

Author(s):

Keiko Umezu ◽

Neal Sugawara ◽

Clark Chen ◽

James E Haber ◽

Richard D Kolodner

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Protein A ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Physical Analysis ◽

Single Stranded Dna ◽

Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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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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