UV-induced Mitotic Recombination Research Articles

Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

Mechanisms of Rad52-Independent Spontaneous and UV-Induced Mitotic Recombination inSaccharomyces cerevisiae

In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His+ gene conversions that have a parental configuration of flanking markers, but approximately 22% of recombinants have associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His+ recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost. We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n - 1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun1's target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.

Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UV-radiation (254 nm) and hyperthermia depend on UV fluence rate

In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic interaction of ultraviolet (UV) light (wavelength, 254 nm) and heat (45–60°C) was studied both for mutagenic and inactivation effects. Simultaneous hyperthermia and UV light treatments increase the frequency of UV-induced mitotic intergenic recombination (crossing-over) and cell inactivation. The enhancing effect was a function of UV light fluence rate. It is concluded that the effect of hyperthermia on low fluence UV or high fluence UV irradiation results in comparable effects on survival and mitotic recombination suggesting similar modulation by hyperthermia of the effects induced by UV at different fluence rates. The interpretation of the data obtained was carried out within the widely accepted point of view considering the synergistic effects as a result of repair ability damage.

The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe.

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1:ura4+ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4- strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4+ strain. Possible explanations are discussed.

Isolation of hem3 mutants from Candida albicans by sequential gene disruption.

Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a approximately 2.0 kb fragment by subcloning and a Bg/II site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the Bg/II site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura+, Leu+ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.

Mapping genes by meiotic and UV-induced mitotic recombination in Coprinus cinereus

Three morphological mutants in Coprinus cinereus—one spontaneous (den-2) and two chemically induced (zigand sta)—were assigned to linkage groups and utilized in meiotic and mitotic mapping. Mutants den-2 and zig belong to linkage group III, den-2 being close to the centromere and about 20 map units (mu) from zig. The mutant sta in linkage group ‘G’ is at a distance of about 37 mu from ade-3. Mitotic mapping confirmed the gene order in linkage group III and provided evidence that trp-2 in linkage group ‘G’ was between the centromere and ade-3. These morphological mutants are compact in colony growth and therefore suited to high-density plating. The rarity of spontaneously occurring mitotic segregants suggests that diploids of Coprinus cinereus, heterozygous for morphoiogical markers in repuision, could serve as useful test systems for rapid screening of chemical mutagen/carcinogens via mitotic recombination studies.

Analysis of non-linearities in frequency curves for UV-induced mitotic recombination in wild-type and excision-repair-deficient strains of yeast

Frequency curves for UV-induced mitotic recombination often are linear at low doses. As dose increases, these curves either increase at higher powers of dose and/or reach a maximum induced frequency and then decline. Similar dose-response patterns have been observed previously for mutation. The non-linearities can arise from higher order effects inherent in the molecular mechanisms of mutagenesis and/or frob ‘δ-effects’ (Eckardt and Haynes, 1977a), i.e., differential probabilities of clone formation for mutant and non-mutant cells. Previously, we have shown that one can distinguish between these two possibilities by plotting the ratio of the induced mutant yield to the linear component of frequency as a function of dose (Haynes et al., 1985). In this study, we have used this ratio, a quantity we call ‘apparent su survival’, to analyse the non-linear regions of the dose-response curves for UV-induced mitotic crossing-over and gene conversion in wild-type ( RAD) and excision-repair-deficient ( rad3) strains of yeast. Plots of apparent survival versus dose reveal the existence of a positive, non-linear component associated with UV-induced gene conversion in RAD, but not rad3, cells. A high dose decline in frequency, which is observed for UV-induced recombination in both strains, can be attributed to δ-effects.

Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of pms1-1 and pms1-2.

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.

Cloning and mapping of the RAD50 gene of Saccharomyces cerevisiae.

The RAD50 gene was cloned as a 4.8 kb fragment in the 2 mu derived plasmid pFL1. The gene resides in a 3.9 kb segment that was subcloned into the plasmid YRp7. The cloned gene complements the deficiency caused by the rad50-1 mutation with respect to gamma-rays, MMS resistance and UV-induced mitotic recombination. Restoration of the Rad+ phenotype occurs when the cloned gene is on a freely replicating multiple-copy plasmid or in the integrated form. Mapping of the cloned gene following integration of the 2 mu plasmid, and of the subclone in plasmid YRp7, showed it to be located on the left arm of chromosome XIV. Tetrad analysis of various crosses involving two different strains carrying rad50-1 showed the mutation to map next to pet2 on chromosome XIV, and not on the right arm of chromosome IV, as previously published.

UV-induced mutation and recombination in yeast: Involvement of an inducible component of repair.

Frequency curves for UV-induced mutation and mitotic recombination are linear at low doses in many strains of S. cerevisiae. At higher doses these curves either increase at higher powers of dose and/or reach a maximum induced frequency and then decline. Previously, we have shown that such dose-response patterns can arise from: (I) non-linearities inherent in the molecular mechanism involved; and (2) "@-effects", i.e. differential probabilities of clone formation for the mutant and non-mutant cells. Non-linearities of the first sort could arise from inducible components of mutagenesis or recombinagenesis, from reductions in the efficiency of error-free repair of from the occurrence of special "two-hit" lesions. " ~-ef~ fects" are associated with expression of the mutant phenotype, or with mutant clone formation. We now have shown that the existence of higher order components of mutagenesis or recombinagenesis is easily determined by examining the ratio of the induced mutant yield to the linear component of mutant frequency as a function of dose. For purely linear responses, and no JJ ~ n -effects , a plot of this ratio (a quantity we call"apparent survival") versus dose generates the actual survival curve of the cells. If the non-linear frequency components arise solely from "@-effects", then the "apparent survival" is the survival curve for the initially induced mutant or recombinant subpopulation. If the "apparent survival" is greater than unity, then there must exist positive, non-linear components associated with the mutational or recombinational processes themselves. Such components exist in RAD wild-type cells undergoing reversion or gene conversion to prototrophy, and are sensitive to inhibition of protein synthesis by cycloheximide. Thus, they may be ascribed to inducible error-prone repair or recombination. Such components do not appear in excision-deficient mutants undergoing forward muation or mitotic crossing over. The high dose decline in frequency, which is observed for forward mutation in RAD and rad2 strains, and for gene conversion and crossing-over in RAD and rad3 diploids, can be attributed to "@-effects". (Supported by NSERC Canada)

A/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

Spontaneous and UV-induced recombination in radiation-sensitive mutants of Schizosaccharomyces pombe.

The rad alleles of 18 unlinked genes of S. pombe were tested for their level of spontaneous meiotic, spontaneous mitotic and UV-induced mitotic recombination in the ade7-50 x ade7-152 interval. The effects of these rad alleles on meiosis and cell morphology were also studied. None of these mutants showed a clear-cut reduction of spontaneous recombination rates, no matter whether they had lost or retained a caffeine-sensitive repair of UV-induced lesions, which has previously been interpreted as a recombinational pathway of DNA repair (Fabre, 1972a; Gentner, 1977; Gentner et al., 1978). rad1-1 was the only mutant with a reduced frequency of UV-induced recombination. Some mutants displayed an increased frequency of mitotic recombination, either spontaneously (rad 15-P, rad 21-45), UV-induced (rad8-190) or both (rad2-44). Previous hypothesis on the contribution of recombination to DNA repair in S. pombe are reconsidered in the light of these data.

UV-induced mitotic recombination and its dependence on photoreactivation and liquid holding in the rad6-1 mutant of Saccharomyces cerevisiae.

Spontaneous and UV-induced mitotic recombination was compared in diploids homozygous for rad6-1 mutation and in the wild-type strain carrying heterozygous markers for detecting gene conversion (hom2-1, hom2-2) and crossing over (ade1, ade2). Diploids homozygous for rad6-1 mutation were characterised by an elevated level of spontaneous and UV-induced mitotic recombination, particularly the intergenic events. Exposure of UV-irradiated strains to visible light resulted in an increased survival and decreased level of mitotic recombination. Liquid holding (LH) differentially affected frequency of mitotic intergenic and intragenic recombination in mutant and wild-type strains, being without any significant effect on cell survival. In a mutant strain intragenic recombination is significantly increased, intergenic only slightly. In the wild-type strain intragenic recombination is slightly decreased but intergenic is not changed by LH. Visible light applied after LH had no effect on survival and mitotic recombination in the wild type, while in the mutant strain photoreactivability of survival was fully preserved and accompanied by a decrease in the frequency of intragenic and intergenic recombination. The results suggest that metabolic pathways responsible for restoring cell survival are independent of or only partly overlapping with those concerning recombination events.

Mitotic recombination in yeast: isolation and characterization of mutants with enhanced spontaneous mitotic gene conversion rates.

Semi-dominant mutants displaying greatly elevated (up to 200-fold above control) levels of spontaneous mitotic recombination have been isolated in a disomic haploid strain of yeast heteroallelic at the arg4 locus. They are designated by the symbol MIC. The mutants variously exhibit associated sensitivity to UV and ionizing radiation and to methyl methanesulfonate, enhanced UV-induced mitotic recombination, and enhanced spontaneous forward mutation rates. Possible enzyme defects and involvement in repair and editing of DNA are discussed. The mutants are expected to simplify the analysis of recombination pathways in yeast.

Genetic characterization of the new morphological and UV-sensitive mutants in Coprinus cinereus. I. A UV-sensitive mutation rad 1 associated with elevated frequencies of mitotic and meiotic recombination.

Studies on the effect of an UV-sensitive mutation, rad 1, in meiotic and mitotic recombination in Coprinus indicated that, in homozygous condition, rad 1 increased the spontaneous meiotic recombination by 50% and UV-induced mitotic intergenic recombination by about 5-fold. The homozygous rad 1 diploid was shown to be much more sensitive to the recombinogenic effects of polyfunctional than than of mono- or non-functional alkylating agents.

Transformation of yeast without the use of foreign DNA

Isolated circular molecules of the yeast plasmid 2-micron DNA were converted to linear molecules with restriction endonuclease PstI and ligated with T4 DNA ligase to PstI restriction fragments of total yeast DNA. A haploid strain of Saccharomyces cerevisiae carrying a deletion in thehis4 locus was transformed with the ligated DNA mixture to histidine prototrophy. One unstable histidine prototrophic transformant was obtained. Grown in the absence of histidine, 70–75% of the cells were auxotrophic and this number increased in non-selective medium. The histidine auxotrophic variants carried a deletion in thehis4 locus which had patterns of complementation and UV-induced mitotic recombination identical to the originalhis4 deletion. When the transformant was crossed to ahis4 strain and sporulated, the unstable histidine prototrophy segregated in a non-mendelian way: All five possible segregations from 0∶4 to 4∶0 were observed. When strains carrying the transformed character were crossed to ahis4 karl strain a low frequency of cytoduction of the unstable histidine prototrophy was observed. Nucleic acid from the transformant was able to transform a strain which carried another deletion in thehis4 locus. Treatment of the transformant with ethidium bromide caused an extensive induction of petites without any observable change in the frequency of histidine prototrophic cells.It is concluded that theHIS4 gene function in the transformant is not stably associated with any chromosome. We take the instability as indication that only one or a few copies of the gene conferring the prototrophy are present in the prototrophic cells. The data are consistent with the assumption that the transformant contains a 2-micron DNA in which is inserted a chromosomal DNA region containing theHIS4 gene. A derivative of the transformant with increased stability has been isolated.

UV-induced mitotic recombination in somatic cells of Drosophila melanogaster

UV-induced mitotic recombination in somatic cells of Drosophila melanogaster

Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis II. A study of repair mechanisms and UV recovery in pyr

Two mutants at the pyr 1 locus have been used to study the radiation sensitivity of pyrimidine auxotrophs of U. maydis. The mutant pyr 1-1 has a reduced level of thymidine nucleotides, and this is a likely basis of the sensitivity. This strain is able to excise pyrimidine dimers from its DNA and is cross-sensitive to γ-rays and nitrosoguanidine (NG) as well as to UV. A diploid heteroallelic at the pyr 1 locus was UV-sensitive but not deficient in UV-induced mitotic recombination. The results suggest that the UV sensitivity may be due to the failure of a repair DNA polymerase to fill post-excision single-strand gaps in the DNA. The mutant pyr 1-1 exhibits the property of UV recovery, and this is shown to be dependent on the presence of dimers in the DNA. A mechanism for UV recovery is proposed in which a repair system, possibly involving recombination, is induced by the UV irradiation.

Radiation-sensitive pyrimidine auxotrophs of Ustilago Maydis II. A study of repair mechanisms and UV recovery in pyr 1

Two mutants at the pyr 1 locus have been used to study the radiation sensitivity of pyrimidine auxotrophs of U. maydis. The mutant pyr 1-1 has a reduced level of thymidine nucleotides, and this is a likely basis of the sensitivity. This strain is able to excise pyrimidine dimers from its DNA and is cross-sensitive to γ-rays and nitrosoguanidine (NG) as well as to UV. A diploid heteroallelic at the pyr 1 locus was UV-sensitive but not deficient in UV-induced mitotic recombination. The results suggest that the UV sensitivity may be due to the failure of a repair DNA polymerase to fill post-excision single-strand gaps in the DNA. The mutant pyr 1-1 exhibits the property of UV recovery, and this is shown to be dependent on the presence of dimers in the DNA. A mechanism for UV recovery is proposed in which a repair system, possibly involving recombination, is induced by the UV irradiation.

Recombination in ultraviolet-sensitive strains of Saccharomyces cerevisiae

Intergenic and intragenic recombination during vegetative growth and at meiosis has been studied by selective plating techniques in diploid strains of yeast homozygous for alleles of six genes governing sensitivity to ultraviolet radiation. The following results were obtained: 1. ( 1) None of the uvs mutations caused a significant increase in spontaneous mitotic intragenic recombination. 2. ( 2)The frequency of intragenic mitotic recombination (gene conversion) induced by UV light was greater among survivors in the sensitive strains compared to wild-type controls, but was less when compared in terms of equal survival. 3. ( 3) The dose-response curves for UV-induced mitotic recombination between the locus of a gene and its centromere were complex, but in most cases the uvs diploids showed a greater response than the controls. 4. ( 4) No differences from the controls were found in the frequency of intragenic or intergenic recombination at meiosis, but significant differences were found between certain uvs strains.