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.     

The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.     

Chromosome dynamics in rad12 mutants of Coprinus cinereus

We have characterized the phenotypes of three rad12 mutants of the basidiomycete Coprinus cinereus, which were isolated on the basis of sensitivity to ionizing radiation. Electron microscopic studies of meiotic nuclear spreads showed that all three rad12 mutants are defective in chromosomal synapsis. For rad12-1 and rad12-4, very limited assembly of the synaptonemal complex occurs. The phenotype of rad12-15 is less severe and longer stretches of synapsed chromosomes are formed. However, for all three alleles mutant nuclei arrest in a diffuse state with little synaptonemal complex structure. Observations made of spreads of acridine orange-stained meiotic nuclei correlated with the electron microscopic data. In rad12 strains, chromosomes condense but do not pair, and they later arrest in a decondensed state; very few rad12 cells enter metaphase I. Homozygous dikaryons of rad12 mutants produce fruiting bodies with significantly fewer basidiospores than are found in wild-type dikaryons. The viability of these spores is greatly reduced: all spores produced by rad12-1 and rad12-4 mushrooms fail to germinate, while only 16% of rad12-15 spores are viable. Recombination within the tract of the ribosomal RNA gene repeats was not significantly different in the mutants when compared with a wild-type congenic control. Quantitative measurements of oidial survival indicate that all three rad12 alleles are sensitive to gamma radiation but insensitive to UV radiation relative to wild-type strains.     

Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.     

Meiosis Can Induce Recombination in rad52 Mutants of SACCHAROMYCES CEREVISIAE

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.     

A similar defect in UV-induced mutagenesis conferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae

The single rad6 and rad18 yeast mutants share a number of physiological and biochemical properties related to DNA repair, suggesting that they affect closely related steps. However, it has been reported that UV-induced mutagenesis is considerably more depressed in rad6 than it is in rad18 cells. In an attempt to better understand the role of these genes, a genetic system believed to differentiate between targeted and untargeted events was used. The data are interpreted to mean that both mutations prevent the occurrence of targeted events, as if they prevent error-prone replication in front of pyrimidine dimers. The number of non-targeted mutants per survivor in each mutant was increased by UV irradiation. This may correspond to a stimulation of the error-prone replication.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

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

Summary 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.     

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.     

Checkpoint controls in Schizosaccharomyces pombe: rad1.

'Checkpoint' controls ensure that the events of the cell cycle are completed in an orderly fashion. For example, such controls delay mitosis until DNA synthesis and repair of radiation-induced DNA damage are complete. The rad series of radiosensitive fission yeast mutants was examined to identify strains deficient for the DNA damage-responsive checkpoint control. Five were identified. A characterization of one (rad1-1) and the wild-type is presented. The rad1-1 mutant does not arrest after irradiation, is sensitive to killing by radiation and is not arrested by hydroxyurea, and thus is also deficient for the DNA synthesis-responsive checkpoint control. The radiosensitivity of the rad1-1 mutant was greatly reduced when irradiated and maintained for 6 h in a non-dividing (density inhibited) state, demonstrating that rad1-1 is repair proficient and radiosensitive only through failure to delay. The checkpoint controls for which rad1 is required appear to regulate G2-M progression through the activity of cdc2, here implicated in this role by the coincidence of the radiation transition point and the cdc2 execution point.     

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.     

Expression of the E.coli ada gene in S.cerevisiae provides cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine in rad6 but not in rad52 mutants.

The Escherichia coli ada gene protein coding region under the control of the yeast alcohol dehydrogenase promoter in the extrachromosomally replicating yeast expression vectors pADHO6C and pVT103LO6C was introduced into the wild-type yeast strains, YNN-27 and FF-18733, and the repair deficient mutants LN-1 (rad1-1), VV-5 (rad6-1), C5-6 (rad52-1) and FF-18742 (rad52::URA3). This resulted in the expression of 3950, 1900, 1870, 1620, 1320 and 1420 fmol ada-encoded ATase/mg protein respectively: transformation with the parent vectors resulted in ATase activities of 3-17 fmol/mg protein. The wild-types, rad1-1 and rad6-1 yeast expressing the bacterial ATase showed increased resistance to the toxic and mutagenic effects of N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). Expression of ATase in the rad52-1 and rad52::URA3 mutants neither complemented their sensitivity, nor reduced the mutagenic effects of this agent. These results suggest that whilst a portion of the toxic and mutagenic lesions induced by MNNG can be repaired in yeast by the E.coli Ada protein in a RAD1- and RAD6-independent manner, the RAD52 gene product may be essential for the complete functioning of the Ada ATase. This is the first suggestion of a possible cofactor requirement for ATase.     

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.     

Indistinguishable Landscapes of Meiotic DNA Breaks in rad50+ and rad50S Strains of Fission Yeast Revealed by a Novel rad50+ Recombination Intermediate

The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein–DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Here, we show that we can detect Rec12-DNA linkages in Sc. pombe rad50⁺ cells, which are proficient for DSB repair. In contrast to the results from Sa. cerevisiae, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50⁺ and rad50S strains of Sc. pombe. These results confirm our earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. We propose that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species—specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species.     

Epistatic Interactions of Radiation-Sensitive (rad) Mutants of CAENORHABDITIS ELEGANS

Six double mutants and a quadruple mutant were derived from four UV radiation-hypersensitive single mutants (rad-1, rad-2, rad-3 and rad-7). Sensitivities of the 11 strains to UV, gamma-radiation and methyl methanesulfonate (MMS) were compared. Of the six double mutants, only the rad-1;rad-2 and rad-3;rad-7 doubles were no more hypersensitive than the most sensitive single mutant to UV-radiation. Thus, rad-1 and rad-2 define one epistasis group, whereas rad-3 and rad-7 define another. Consistent with this model was the observation that rad-1 and rad-2, but not rad-3 and rad-7, were hypersensitive to gamma-radiation. In addition, none of the multiple mutants was more hypersensitive to gamma-radiation than the most sensitive single rad mutant. No synergistic interactions of the rad mutations with respect to MMS sensitivities were observed.     

Cloning and characterisation of the Schizosaccharomyces pombe rad8 gene, a member of the SNF2 helicase family.

The Schizosaccharomyces pombe rad8 mutant is sensitive to both UV and gamma irradiation. We have cloned the rad8 gene by complementation of the UV sensitivity of a rad8.190 mutant strain. The gene comprises an open reading frame of 3.4 kb which does not contain any introns and is capable of encoding a 1133 amino acid protein of 129 kDa. Deletion of the gene indicates that it is not essential for cell viability. Recognisable motifs are present for a nuclear localisation signal, a RING finger and helicase domains. The predicted protein is a member of the SNF2 subfamily of proteins and shows particular homology to the Saccharomyces cerevisiae RAD5 protein. Double mutant analysis demonstrated that the rad8 mutant is not epistatic to mutants in the excision repair pathway (rad13) or checkpoint pathway (rad9). Analysis of radiation sensitivity though the cell cycle indicates that, unlike most other rad mutants, rad8 is most sensitive to irradiation during the G1/S period.     

Identification of rad27 Mutations That Confer Differential Defects in Mutation Avoidance, Repeat Tract Instability, and Flap Cleavage

In eukaryotes, the nuclease activity of Rad27p (Fen1p) is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments. Genetic analysis of Saccharomyces cerevisiae also has identified a role for Rad27p in mutation avoidance. rad27Delta mutants display both a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result primarily from duplications of DNA sequences that are flanked by direct repeats. These observations suggested that Rad27p activities in DNA replication and repair could be altered by mutagenesis and specifically assayed. To test this idea, we analyzed two rad27 alleles, rad27-G67S and rad27-G240D, that were identified in a screen for mutants that displayed repeat tract instability and mutator phenotypes. In chromosome stability assays, rad27-G67S strains displayed a higher frequency of repeat tract instabilities relative to CAN1 duplication events; in contrast, the rad27-G240D strains displayed the opposite phenotype. In biochemical assays, rad27-G67Sp displayed a weak exonuclease activity but significant single- and double-flap endonuclease activities. In contrast, rad27-G240Dp displayed a significant double-flap endonuclease activity but was devoid of exonuclease activity and showed only a weak single-flap endonuclease activity. Based on these observations, we hypothesize that the rad27-G67S mutant phenotypes resulted largely from specific defects in nuclease function that are important for degrading bubble intermediates, which can lead to DNA slippage events. The rad27-G240D mutant phenotypes were more difficult to reconcile to a specific biochemical defect, suggesting a structural role for Rad27p in DNA replication and repair. Since the mutants provide the means to relate nuclease functions in vitro to genetic characteristics in vivo, they are valuable tools for further analyses of the diverse biological roles of Rad27p.     

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.