Transformation and recombination in rad mutants of Saccharomyces cerevisiae

Summary Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single-and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trpl-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trpl-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura352 mutant locus where only integration events have been observed, and at the trpl-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

Genome instability in rad54 mutants of Saccharomyces cerevisiae

The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair. Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells. Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation. The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication. Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays. Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog. In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells. Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss.     

Defective thymine dimer excision in radiation-sensitive mutants rad10 and rad16 of Saccharomyces cerevisiae.

Two rad mutants of yeast, rad10 and rad16, are shown to be defective in the removal of UV-induced pyrimidine dimers since DNAs obtained from irradiated cells following a post-irradiation incubation in the dark still retain UV-endonuclease-sensitive sites. Both rad10 and rad16 mutants are in the same pathway of excision-repair as the rad1, rad2, rad3 and rad4 mutants.     

Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae

Summary Excision of pyrimidine dimers and interstrand DNA crosslinks was examined in the deletion mutants rad7-1, rad23-1, and rad7-1 rad23-1. These mutants remove pyrimidine dimers and crosslinks much less efficiently than the RAD ⁺ strains; only 30–60% of pyrimidine dimers and 25–40% of crosslinks are removed even after prolonged incubation. The rad7 and rad23 mutations may represent defects in protein factors which increase the efficiency of the nicking enzyme complex or make chromatin more accessible to the nicking activity.     

Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae.

A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity.     

Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 UV-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.     

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Summary Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (radl-1, 2, 3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants radl-1, 3, 5, 6, 9 and 11 differ in their response to LH afterr the two treatments. Survival of radl-1 and rad3 increases significantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11 and 22 show marked LHR after UV but no increase of survival after DEB treatment.     

Deletion analysis of the SUP2 gene in Saccharomyces cerevisiae

The sup2 mutations of the yeast Saccharomyces cerevisiae or plasmid-mediated amplification of the wild type SUP2 gene lead to suppression of different types of nonsense mutations. The Sup2 protein includes a C-terminal region homologous to elongation factor EF-1 alpha and an unique N-terminal region. The SUP2 is an essential gene. The functional role of different regions of the SUP2 gene was investigated, by deleting them without disruption of the reading frame. Such constructs were maintained in yeast on episomal or centromeric plasmids. It was shown that the region, homologous to EF-1 alpha is necessary for viability, while the remaining N-terminal part is nonessential. The region of the first 154 amino acids is necessary and sufficient for the suppressor effect, caused by plasmid-mediated amplification of the SUP2 gene.     

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. This breakup of DNA in rad18-2 cells is not degradation due to cell death since wild type cells irradiated to similar low survival levels do not show this breakup of DNA with 8 h incubation. The breakup of DNA in rad18-2 cells is not due to replication gaps being formed by synthesis on a damaged template since treatment of rad18-2 a mating type cells with alpha factor, to prevent initiation of DNA synthesis, does not prevent breakup of the DNA.     

Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae

A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model for RAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.     

Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Delta mutants are defective in all the G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine(115) residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24(K115R) fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G(1)-, S- and G(2)/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.     

Meiotic recombination in RAD54 mutants of Saccharomyces cerevisiae

The Rad54 protein is an important component of the recombinational DNA repair pathway in vegetative Saccharomyces cerevisiae cells. Unlike those in other members of the RAD52 group, the meiotic defect in rad54 is rather mild, reducing spore viability only to 26%–65%. A consistently greater requirement for Rad54p during meiosis was observed in hybrid strains, suggesting that Rad54p has a certain role in interhomolog interactions. Such a role is probably minor as no recombination defects were found in the surviving gametes in three genetic intervals on chromosome V. Also, the spore viability pattern in tetrads did not reflect an increase in nondisjunction at meiosis I indicative of a meiotic recombination defect. We suggest that the meiotic defect of rad54 cells lies in the failure to repair meiosis-specific double-strand breaks outside the context of the highly differentiated pathway leading to interhomolog joint molecules and meiotic crossovers that ensure accurate segregation at meiosis I. Received: 15 November 1999; in revised form: 11 January 2000 / Accepted: 11 January 2000     

A complex pattern of sensitivity to simple monofunctional alkylating agents exists amongst the rad mutants of Saccharomyces cerevisiae

Summary The radiation-sensitive rad mutants of the yeast Saccharomyces cerevisiae exhibit a complex pattern of sensitivity to simple monofunctional alkylating agents. The RAD1, RAD2, RAD4 and RAD14 genes of the RAD3 epistasis group are implicated in the repair of ethylations to DNA. The RAD3, RAD10 and RAD16 genes of this group are not involved. The RAD4 and RAD14 genes have a particular role in repair following exposure to those ethylating agents that preferentially alkylate oxygen, but not to those that preferentially ethylate nitrogen. The RAD1 and RAD2 genes are involved in the repair of damage induced by all the ethylating agents used except EMS. The mutants in this group that are sensitive to ENU were not sensitive to MNU, suggesting that nucleotide excision operates on ethylations but not on methylations.In the RAD6 group, the RAD6 and RAD18 genes are involved in DNA repair after exposure to all the alkylating agents tested, whereas RAD8 appears to have a role in the repair of O-alkylations but not N-alkylations. RAD9 operates in the repair of methylations and ethylations, but does not influence events after exposure to EMS. In the RAD52 group, the mutants tested were sensitive to ENU and DES. Thus some members of all three epistasis groups are involved in the repair of alkylations to DNA.Abbreviations DES diethylsulphate - EMS ethylmethanesulphonate - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - ENU N-ethylnitrosourea - MNU N-methylnitrosourea - DMSO dimethylsulphoxide - MMS methylmethanesulphonate