The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair.

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, delta and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae.

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase delta, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase delta and a deletion in the REV3 gene had a complete repair defect, both at permissive (23 degrees C) and restrictive (38 degrees C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants.

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae.

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase delta on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37 degrees C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys(-)-->Lys+ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23 degrees C or 37 degrees C.

DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae.

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 degrees C), but is not affected at the permissive temperature (23 degrees C).

Role of the CDC8 gene in the repair of single strand breaks in DNA of the yeast Saccharomyces cerevisiae.

It has been found that the repair of single strand breaks is defective in the DNA replication mutants cdc8-1 and cdc8-3 of Saccharomyces cerevisiae both in permissive (23 degrees C) and restrictive conditions (36 degrees C). In permissive conditions we observed a significant delay in single strand break repair in a diploid strain HB7 (cdc8-1/cdc8-1), as compared with the wild-type strain. Under restrictive conditions no repair was observed, but rather degradation of MMS-damaged DNA occurred.

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers.

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E.

Repair of UV-irradiated plasmid DNA in a Saccharomyces cerevisiae rad3 mutant deficient in excision-repair of pyrimidine dimers.

The repair of UV-irradiated DNA of plasmid pBB29 was studied in an incision-defective rad3-2 strain of Saccharomyces cerevisiae and in a uvrA6 strain of Escherichia coli by the measurement of cell transformation. Plasmid pBB29 used in these experiments contained as markers the DNA of nuclear yeast gene LEU-2 and DNA of the bacterial plasmid pBR327 with resistance to Tet and Amp enabling simultaneous screening of transformant cells in both microorganisms. We found that the yeast rad3-2 mutant, deficient in incision of UV-induced pyrimidine dimers in nuclear DNA, was fully capable of repairing such lesions in plasmid DNA.

Repair of gamma-ray induced DNA strand breaks in the radiation-sensitive mutant rad18-2 of Saccharomyces cerevisiae.

The repair of gamma-ray induced DNA single and double-strand breaks was looked at in wild type and rad18-2 strains of the yeast Saccharomyces cerevisiae using sucrose gradient centrifugation. It was found that rad18-2 diploid cells could repair single and double-strand breaks induced by gamma-rays. It was also found that rad18-2 cells experienced a breakup of their DNA during post-irradiation incubation to a size smaller than seen in cells just receiving irradiation.

Repair of interstrand cross-links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: the RAD3 system and the RAD51 system.

We have studied the role of the excision-repair system and the recombination-repair system in the removal of cross-links and monoadducts caused by furocoumarins plus 360 nm radiation in yeast DNA by neutral and alkaline sucrose gradients and by a fluorometric procedure which detects cross-linked DNA molecules. We found that the excision-repair system, represented by the rad3 mutations, is required both for the removal of monoadducts, causing single-strand break formation, and for the removal of cross-links, causing double-strand break formation. The recombination-repair system, represented by the rad51 mutation, is necessary for double-strand break repair following cross-link removal, but it has no role in the repair of monoadducts.

Relation between liquid-holding recovery, DNA repair, and mitotic recombination in the rad3 mutant of Saccharomyces cerevisiae after treatment with diepoxybutane (DEB).

The rad3 mutant is characterized by a high level of liquid-holding recovery after DEB treatment. The recovery is abolished when the treated cells are postincubated in growth medium, but the effect can be cancelled by suppression of DNA and protein synthesis by specific inhibitors. Alkaline sucrose gradient sedimentation revealed that DEB induces single strand breaks in DNA which are not repaired during post-treatment incubation in growth medium or during LH.

Repair of MMS-induced DNA double-strand breaks in haploid cells of Saccharomyces cerevisiae, which requires the presence of a duplicate genome.

The formation and repair of double-strand breaks induced in DNA by MMS was studied in haploid wild type and MMS-sensitive rad6 mutant strains of Saccharomyces cerevisiae with the use of the neutral and alkaline sucrose sedimentation technique. A similar decrease in average molecular weight of double-stranded DNA from 5--6 X 10(8) to 1--0.7 X 10(8) daltons was observed following treatment with 0.

Endonuclease for apurinic sites in yeast. Comparison of the enzyme activity in the wild type and in rad mutants of Saccharomyces cerevisiae to MNS.

It was found that yeast cells contain an endonuclease specific for apurinic sites in DNA which has no effect on DNA with normal strands or on strands with alkylated sites. The enzyme activity was studied in the RAD strain and in rad 6, rad 18-2 and rad 21 mutants, all very sensitive to MMS, as compared to the wild type. The level of endonuclease activity does not differ much between the tested strains, regardless of their differences in susceptibility to MMS.

Alkaline sucrose sedimentation studies of MMS-induced DNA single-strand breakage and rejoining in the wild type and in UV-sensitive mutants of Saccharomyces cerevisiae.

MMS-induced DNA single-strand breakage and rejoining was studied in the RAD strain and in rad6 and rad21 mutants, both very sensitive to this treatment as compared with the wild type. Alkaline sucrose gradient centrifugation showed that MMS treatment reduced the molecular weight of DNA in the RAD strain and in rad6 and rad21 mutants to the same extent. Four hours of post-incubation in synthetic growth medium after treatment with a dose of 0.

Manganese mutagenesis in yeast. VI. Mn2+ uptake, mitDNA replication and ER induction: comparison with other divalent cations.

A medium was found in which manganese efficiently induces erythromycin-resistant mitochondrial mutations, and which is suitable for measuring Mn2+ uptake and the labelling of DNA (fig. 1). Mn2+ uptake is stimulated by glucose and slowed down by cycloheximide (Fig 2).