Genetic and biochemical consequences of thymidylate stress

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.     

DNA repair and the genetic effects of thymidylate stress in yeast

Folate antagonists, such as aminopterin, methotrexate and various sulfonamides, block de novo thymidylate biosynthesis in Saccharomyces cerevisiae. The resulting starvation for thymine nucleotides is lethal and recombinagenic in RAD wild-type strains. In this paper we report our studies of these effects in repair-deficient yeast. Antifolate treatment of various rad mutants revealed that repair defects influence the killing and recombination caused by thymidylate deprivation. Compared to a RAD wild-type strain, diploids homozygous for rad3, rad6 or rad18 were more resistant to cell killing. Thus, contrary to findings with conventional DNA-damaging agents, the lethal effects of thymidylate starvation appear to be ameliorated by certain DNA repair deficiencies. On the other hand, a rad50 strain was extremely sensitive to the antifolates. Within this series of diploids, increasing sensitivity to thymidylate starvation was accompanied by an increase in recombination frequencies. The degrees of lethality and recombination, induced by thymidylate depletion, were correlated with the severity of DNA-strand breakage in the RAD and rad50 strains. Experiments with diploids homozygous for rad52, rad54 or rad57 suggested that aborted recombination events, provoked by thymidylate deprivation, caused chromosome loss. Furthermore, the repair defects in these mutants indicated that double-strand breaks are among the lethal lesions induced by thymine nucleotide starvation. Finally, we discuss the possibility that the recombinagenicity of thymidylate stress may account for one type of acquired resistance to methotrexate in mammalian cells.     

RAD52-Independent Mitotic Gene Conversion in SACCHAROMYCES CEREVISIAE Frequently Results in Chromosomal Loss

We have examined spontaneous, interchromosomal mitotic recombination events between his4 alleles in both Rad+ and rad52 strains of Saccharomyces cerevisiae. In Rad+ strains, 74% of the His+ prototrophs resulted from gene conversion events without exchange of flanking markers. In diploids homozygous for the rad52-1 mutation, the frequency of His+ prototroph formation was less than 5% of the wild-type value, and more than 80% of the gene conversion events were accompanied by an exchange of flanking markers. Most of the rad52 intragenic recombination events arose by gene conversion accompanied by an exchange of flanking markers and not by a simple reciprocal exchange between the his4A and his4C alleles. There were also profound effects on the kinds of recombinant products that were recovered. The most striking effect was that RAD52-independent mitotic recombination frequently results in the loss of one of the two chromosomes participating in the gene conversion event.     

The relationship between homology length and crossing over during the repair of a broken chromosome

Homologous recombination can result in the transfer of genetic information from one DNA molecule to another (gene conversion). These events are often accompanied by a reciprocal exchange between the interacting molecules (termed "crossing over"). This association suggests that the two types of events could be mechanistically related. We have analyzed the repair, by homologous recombination, of a broken chromosome in yeast. We show that gene conversion can be uncoupled from crossing over when the length of homology of the interacting substrates is below a certain threshold. In addition, a minimal length of homology on each broken chromosomal arm is needed for crossing over. We also show that the coupling between gene conversion and crossing over is affected by the mismatch repair system; mutations in the MSH2 or MSH6 genes cause an increase in the crossing over observed for short alleles. Our results provide a mechanism to explain how chromosomal recombinational repair can take place without altering the stability of the genome.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.     

A plasmid model to study genetic recombination in yeast

Chimeric plasmids have been constructed containing two heteroallelic mutant copies of the yeast HIS3 gene as an inverted repetition. Intramolecular exchange events between these two allelic mutant copies are capable of generating a wild-type allele. Plasmids containing two mutant heteroalleles have been transformed into appropriate his3^? yeast strains, and the frequency of exchange events generating His⁺ prototrophs has been measured during mitotic division. After 20 generations of growth under nonselective conditions, between 0.1 and 1 % of the transformed yeast cells become His⁺ prototrophs. This percentage decreases at least ten-fold in a strain with a rad52 mutation. Plasmid molecules having undergone exchange events have been isolated from yeast cells and have been examined after transfer to Escherichia coli. Physical examination shows that less than 10 % of the plasmids having undergone genetic exchange have also undergone an internal reciprocal recombination event as evidenced by reorientation of linked restriction sites. The remainder of the plasmids having undergone genetic exchange do not exhibit reciprocal recombination. Characterization of the individual allelic copies within a plasmid having undergone exchange reveals that in 24 of 25 examples only one of the two HIS3 copies has become wild type, and that either copy is equally likely to become wild type. We conclude that the model plasmid we have constructed undergoes intramolecular genetic exchange events and will be useful for studying genetic recombination.     

Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.     

Association of reciprocal exchange with gene conversion between the repeated segments of 2-micron circle

The occurrence of reciprocal exchange of flanking DNA during gene conversion between the repeated segments of the yeast plasmid, 2-micron circle has been examined. The conversion event is induced by making a double-stranded gap within one of the repeats in vitro and allowing the gap to be repaired in vivo. The repair takes place with frequent recombination of flanking markers. Neither the topology of the plasmid substrates (linear or circular) nor the relative orientation of the repeats affects the association rule significantly. These events are reminiscent of meiotic gene conversion between homologous chromosomes but contrast sharply with mitotic or meiotic intrachromosomal gene conversion. It would appear that the difference between the outcomes of intramolecular gene conversion on a chromosome and on a plasmid gapped in vitro does not result from the different physical states of intracellular versus transformed DNA. A gene conversion event in a 2-micron circle : : Tn5 plasmid mediated by the 2-micron circle recombinase (FLP) in vivo, which is formally analogous to the yeast mating type interconversion, often results in recombination of flanking markers. The reaction can be mimicked, in the absence of FLP, by gapping the plasmid within one of the 2-micron circle repeats in vitro and carrying out gap repair in vivo.     

Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae.

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.     

Evidence for the murine IgH mu locus acting as a hot spot for intrachromosomal homologous recombination

Homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. It can occur either by gene conversion, a process of unidirectional genetic exchange, or by reciprocal crossing over. Homologous recombination is well known for its role in generating genetic diversity in meiosis and, in mitosis, as a DNA repair mechanism. In the immune system, the evidence suggests a role for homologous recombination in Ig gene evolution and in the diversification of Ab function. Previously, we reported the occurrence of homologous recombination between repeated, donor and recipient alleles of the Ig H chain mu gene C (Cmu) region residing at the Ig mu locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cmu region heteroalleles to learn more about the intrachromosomal homologous recombination process. A high frequency of homologous recombination (gene conversion) was observed for markers spanning the entire recipient Cmu region, suggesting that recombination might initiate at random sites within the Cmu region. The Cmu region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cmu heteroalleles were tested for recombination in ectopic genomic positions, the mean frequency of gene conversion was reduced by at least 65-fold. These results are consistent with the murine IgH mu locus behaving as a hot spot for intrachromosomal homologous recombination.     

Analysis of the Mechanism for Reversion of a Disrupted Gene

A positive selection system for intrachromosomal recombination in Saccharomyces cerevisiae has been developed. This was achieved by integration of a plasmid containing an internal fragment of the HIS3 gene into its chromosomal location. This resulted in two copies of the HIS3 gene one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end. Reversion of the gene disruption could be brought about by plasmid excision, unequal sister chromatid exchange or sister chromatid conversion. The purpose of this study was to define the mechanisms involved in reversion of the gene disruption. The frequency of plasmid excision could be determined by placing a yeast sequence bearing an origin of replication onto the plasmid that was subsequently integrated into the yeast genome. Unequal sister chromatid exchange and conversion could be distinguished by determining the nature of the reciprocal product by Southern blotting. The results indicate that reversion might occur mainly by conversion between sister chromatids. This is because the frequency of plasmid excision was about two orders of magnitude lower than the overall frequency of reversion and no reciprocal product indicative of sister chromatid exchange was found. The findings of this presentation suggest that conversion might be an important mechanism for recombination of sister chromatids and possibly for repair of damaged DNA in S or G2.     

A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination.

We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.     

Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Recombination in Aspergillus

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.     

Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces 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.     

Meiosis in Asynaptic Yeast

The Saccharomyces cerevisiae red1 mutant fails to assemble synaptonemal complex during meiotic prophase. This mutant displays locus-specific reductions in interchromosomal gene conversion and a moderate reduction in crossing over. The occurrence of a significant amount of meiotically induced recombination in the red1 mutant indicates that the synaptonemal complex is not absolutely required for meiotic exchange. The RED1 gene product is required for intrachromosomal recombination in some assays but not others. Chromosomes that have undergone reciprocal exchange nevertheless nondisjoin in red1 mutants, indicating that crossovers are not sufficient for disjunction. Epistasis studies reveal that HOP1 is epistatic to RED1, and that RED1 acts in an independent pathway from MER1. A model for the function of the RED1 gene product in chromosome synapsis is discussed.     

Long,interrupted conversion tracts initiated by cog in Neurospora crassa.

Multiple polymorphisms distinguish Emerson and Lindegren strains of Neurospora crassa within the histidine-3 gene and in its distal flank. Restriction site and sequence length polymorphism in a set of 14 PCR products covering this 6.9-kb region were used to identify the parental origin of DNA sequence information in prototrophic progeny of crosses heterozygous for auxotrophic mutations in his-3 and the silent sequence differences. Forty-one percent of conversion tracts are interrupted. Where the absence of rec-2+ permits activity of the recombination hotspot cog, conversion appears to originate at cog and conversion tracts are up to 5.9 kb long. The chromosome bearing cog(L), the dominant allele that confers a high frequency of recombination, is almost invariably the recipient of information. In progeny from crosses heterozygous rec-2/rec-2+, conversion tracts are much shorter, most are not initiated at cog and either chromosome seems equally likely to be converted. Although 32% of his-3 prototrophs have a crossover that may be associated with conversion, it is suggested that the apparent association between conversion and crossing over at this locus may be due to confounding of coincidental events rather than to a mechanistic relationship.     

Gene Conversion Alone Accounts for More than 90% of Recombination Events at the Am Locus of Neurospora Crassa

We have used closely flanking molecular markers located ~4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am(1) am(6). Ninety-three percent of prototrophs were generated by events that did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (~0.07) than estimated previously using more distant flanking markers. This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favor of retaining the parental association of flanking sequences. Because estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (~0.35), our observation may have general significance.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences

The centromere of chromosome III (CEN3) of yeast has been examined for its ability to inhibit meiotic recombination in adjacent sequences. The effect of the centromere was investigated when it was adjacent to both of the recombining sequences (homozygous) or adjacent to only one of the two recombining DNA segments (hemizygous). When homozygous, CEN3 exerts a bidirectional repression of crossing over and a strong inhibition of gene conversion. This suggests that CEN3 reduces the frequency of crossing over by interfering with the initiation of proximal recombination events. When hemizygous, CEN3 impairs the ability of adjacent sequences to act as the recipient of genetic information during gene conversion. These results support the idea that the initiating event in yeast meiotic recombination involves the recipient molecule.