The new yeast is a mouse.

Recombination hotspots are determined not only by features of the local genome but also by sequences acting at a considerable distance both in cis and trans.

DNA damage: from causes to cures.

In order to maintain genome integrity, it is essential that any DNA damage is repaired. This is achieved in diverse ways in all cells to ensure cellular survival. There is a large repertoire of proteins that remove and repair DNA damage. However, sometimes these processes do not function correctly, leading to genome instability. Studies of DNA repair and genome instability and their causes and cures were showcased in the 2008 Biochemical Society Annual Symposium. The present article provides a summary of the talks given and the subsequent papers in this issue of Biochemical Society Transactions.

Interaction of genetic and environmental factors in Saccharomyces cerevisiae meiosis: the devil is in the details.

One of the most important principles of scientific endeavour is that the results be reproducible from lab to lab. Although research groups rarely redo the published experiments of their colleagues, research plans almost always rely on the work of someone else. The assumption is that if the same experiment were repeated in another lab, results would be so similar that the same interpretation would be favoured. This notion allows one researcher to compare his/her own results to earlier work from other labs. An essential prerequisite for this is that the experiments are done in identical conditions and therefore the methodology must be clearly stated. While this may be scientific common sense, adherence is difficult because "standard" methods vary from one laboratory to another in subtle ways that are often not reported. More importantly, for many years the field ofyeast meiotic recombination considered typical differences to be innocuous. This chapter will highlight the documented environmental and genetic variables that are known to influence meiotic recombination in Saccharomyces cerevisiae. Other potential methodological sources of variation in meiotic experiments are also discussed. A careful assessment of the effects of these variables, has led to insights into our understanding of the control of recombination and meiosis.

Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase.

The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIalpha to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an 'anti-recombinase'.

Why molecular chaperones buffer mutational damage: a case study with a yeast Hsp40/70 system.

The malfunctioning of molecular chaperones may result in uncovering genetic variation. The molecular basis of this phenomenon remains largely unknown. Chaperones rescue proteins unfolded by environmental stresses and therefore they might also help to stabilize mutated proteins and thus mask damages. To test this hypothesis, we carried out a genomewide mutagenesis followed by a screen for mutations that were synthetically harmful when the RAC-Ssb1/2 cytosolic chaperones were inactive. Mutants with such a phenotype were found and mapped to single nucleotide substitutions. However, neither the genes identified nor the nature of genetic lesions implied that folding of the mutated proteins was being supported by the chaperones. In a second screen, we identified temperature-sensitive (ts) mutants, a phenotype indicative of structural instability of proteins. We tested these for an association with sensitivity to loss of chaperone activity but found no such correlation as might have been expected if the chaperones assisted the folding of mutant proteins. Thus, molecular chaperones can mask the negative effects of mutations but the mechanism of such buffering need not be direct. A plausible role of chaperones is to stabilize genetic networks, thus making them more tolerant to malfunctioning of their constituents.

Trans events associated with crossovers are revealed in the absence of mismatch repair genes in Saccharomyces cerevisiae.

Genetic analysis of recombination in Saccharomyces cerevisiae has revealed products with structures not predicted by the double-strand break repair model of meiotic recombination. A particular type of recombinant containing trans heteroduplex DNA has been observed at two loci. Trans events were originally identified only in tetrads in which the non-Mendelian segregations were not associated with a crossover. Because of this, these events were proposed to have arisen from the unwinding of double Holliday junctions. Previous studies used palindromes, refractory to mismatch repair, as genetic markers whereas we have used a complementary approach of deleting mismatch repair proteins to identify heteroduplex DNA. We found that the markers occurred in trans and were associated with crossovers. In both mlh1Delta and msh2Delta strains, the frequency of trans events associated with a crossover exceeded that predicted from the random association of crossovers with noncrossover trans events. We propose two different models to account for trans events associated with crossovers and discuss the relevance to wild-type DSB repair.

MLH1 and MSH2 promote the symmetry of double-strand break repair events at the HIS4 hotspot in Saccharomyces cerevisiae.

Double-strand breaks (DSBs) initiate meiotic recombination. The DSB repair model predicts that both genetic markers spanning the DSB should be included in heteroduplex DNA and be detectable as non-Mendelian segregations (NMS). In experiments testing this, a significant fraction of events do not conform to this prediction, as only one of the markers displays NMS (one-sided events). Two explanations have been proposed to account for the discrepancies between the predictions and experimental observations. One suggests that two-sided events are the norm but are "hidden" as heteroduplex repair frequently restores the parental configuration of one of the markers. Another explanation posits that one-sided events reflect events in which heteroduplex is formed predominantly on only one side of the DSB. In the absence of heteroduplex repair, the first model predicts that two-sided events would be revealed at the expense of one-sided events, while the second predicts no effect on the distribution of events when heteroduplex repair is lost. We tested these predictions by deleting the DNA mismatch repair genes MSH2 or MLH1 and analyzing the proportion of two-sided events. Unexpectedly, the results do not match the predictions of either model. In both mlh1Delta and msh2Delta, the proportion of two-sided events is significantly decreased relative to wild type. These observations can be explained in one of two ways. Either Msh2p/Mlh1p-independent mispair removal leads to restoration of one of the markers flanking the DSB site or Msh2p/Mlh1p actively promote two-sided events.

Apparent Epigenetic Meiotic Double-Strand-Break Disparity in Saccharomyces cerevisiae: A Meta-Analysis.

Previously published, and some unpublished, tetrad data from budding yeast (Saccharomyces cerevisiae) are analyzed for disparity in gene conversion, in which one allele is more often favored than the other (conversion disparity). One such disparity, characteristic of a bias in the frequencies of meiotic double-strand DNA breaks at the hotspot near the His4 locus, is found in diploids that undergo meiosis soon after their formation, but not in diploids that have been cloned and frozen. Altered meiotic DNA breakability associated with altered metabolism-related chromatin states has been previously reported. However, the above observations imply that such differing parental chromatin states can persist through at least one chromosome replication, and probably more, in a common environment. This conclusion may have implications for interpreting changes in allele frequencies in populations.

MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae.

To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility.

Does crossover interference count in Saccharomyces cerevisiae?

We previously proposed a "counting model" for meiotic crossover interference, in which double-strand breaks occur independently and a fixed number of noncrossovers occur between neighboring crossovers. Whereas in some organisms (group I) this simple model alone describes the crossover distribution, in other organisms (group II) an additional assumption--that some crossovers lack interference--improves the fit. Other differences exist between the groups: Group II needs double-strand breaks and some repair functions to achieve synapsis, while repair in group I generally occurs after synapsis is achieved; group II, but not group I, has recombination proteins Dmc1, Mnd1, and Hop2. Here we report experiments in msh4 mutants that are designed to test predictions of the revised model in a group II organism. Further, we interpret these experiments, the above-mentioned differences between group I and II meiosis, and other data to yield the following proposal: Group II organisms use the repair of leptotene breaks to promote synapsis by generating double-Holliday-junction intermediates that lock homologs together (pairing pathway). The possible crossover or noncrossover resolution products of these structures lack interference. In contrast, for both group I and group II, repair during pachytene (disjunction pathway) is associated with interference and generates only two resolution types, whose structures suggest that the Holliday junctions of the repair intermediates are unligated. A crossover arises when such an intermediate is stabilized by a protein that prevents its default resolution to a noncrossover. The protein-binding pattern required for interference depends on clustering of sites that have received, or are normally about to receive, meiotic double-strand breaks.

Epistasis and hybrid sterility in Saccharomyces.

Hybrid sterility is thought to be due to deleterious epistatic interactions between genes from different species. Here we demonstrate that dominant genic incompatibility does not contribute to sterility in hybrids between Saccharomyces cerevisiae and five closely related species. Sterile diploids were made fertile by genome doubling to produce hybrid tetraploids. Based on these and previous results, we conclude that neither genic incompatibility nor classical chromosomal speciation models apply.

Separable roles for Exonuclease I in meiotic DNA double-strand break repair.

Exo1 is a member of the Rad2 protein family and possesses both 5'-3' exonuclease and 5' flap endonuclease activities. In addition to performing a variety of functions during mitotic growth, Exo1 is also important for the production of crossovers during meiosis. However, its precise molecular role has remained ambiguous and several models have been proposed to account for the crossover deficit observed in its absence. Here, we present physical evidence that the nuclease activity of Exo1 is essential for normal 5'-3' resection at the Spo11-dependent HIS4 hotspot in otherwise wild-type cells. This same activity was also required for normal levels of gene conversion at the locus. However, gene conversions were frequently observed at a distance beyond that at which resection was readily detectable arguing that it is not the extent of the initial DNA end resection that limits heteroduplex formation. In addition to these nuclease-dependent functions, we found that an exo1-D173A mutant defective in nuclease activity is able to maintain crossing-over at wild-type levels in a number of genetic intervals, suggesting that Exo1 also plays a nuclease-independent role in crossover promotion.

Distinct regulation of Mlh1p heterodimers in meiosis and mitosis in Saccharomyces cerevisiae.

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.

The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance.

BACKGROUND: The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination) and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of 'second strand capture' when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent) are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures. CONCLUSIONS: This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout meiosis.

Temperature-dependent modulation of chromosome segregation in msh4 mutants of budding yeast.

BACKGROUND: In many organisms, homologous chromosomes rely upon recombination-mediated linkages, termed crossovers, to promote their accurate segregation at meiosis I. In budding yeast, the evolutionarily conserved mismatch-repair paralogues, Msh4 and Msh5, promote crossover formation in conjunction with several other proteins, collectively termed the Synapsis Initiation Complex (SIC) proteins or 'ZMM's (Zip1-Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3). zmm mutants show decreased levels of crossovers and increased chromosome missegregation, which is thought to cause decreased spore viability. PRINCIPAL FINDINGS: In contrast to other ZMM mutants, msh4 and msh5 mutants show improved spore viability and chromosome segregation in response to elevated temperature (23 degrees C versus 33 degrees C). Crossover frequencies in the population of viable spores in msh4 and msh5 mutants are similar at both temperatures, suggesting that temperature-mediated chromosome segregation does not occur by increasing crossover frequencies. Furthermore, meiotic progression defects at elevated temperature do not select for a subpopulation of cells with improved segregation. Instead, another ZMM protein, Zip1, is important for the temperature-dependent improvement in spore viability. CONCLUSIONS: Our data demonstrate interactions between genetic (zmm status) and environmental factors in determining chromosome segregation.

Hybrid speciation in experimental populations of yeast.

Most models of speciation require gradual change and geographic or ecological isolation for new species to arise. Homoploid hybrid speciation occurred readily between Saccharomyces cerevisiae and Saccharomyces paradoxus. Hybrids had high self-fertility (about 82%), low fertility when backcrossed to either parental species (about 7.5%), and vigorous growth under different thermal environments that favored one or the other of the parental species. Extensive karyotypic changes (tetrasomy) were observed in the hybrids, although genic incompatibilities accounted for 50% of the variation in self-fertility.

Small fitness effects and weak genetic interactions between deleterious mutations in heterozygous loci of the yeast Saccharomyces cerevisiae.

Rare, random mutations were induced in budding yeast by ethyl methanesulfonate (EMS). Clones known to bear a single non-neutral mutation were used to obtain mutant heterozygotes and mutant homozygotes that were later compared with wild-type homozygotes. The average homozygous effect of mutation was an approximately 2% decrease in the growth rate. In heterozygotes, the harmful effect of these relatively mild mutations was reduced approximately fivefold. In a test of epistasis, two heterozygous mutant loci were paired at random. Fitness of the double mutants was best explained by multiplicative action of effects at single loci, with little evidence for epistasis and essentially excluding synergism. In other experiments, the same mutations in haploid and heterozygous diploid clones were compared. Regardless of the haploid phenotypes, mildly deleterious or lethal, fitness of the heterozygotes was decreased by less than half a per cent on average. In general, the results presented here suggest that most mutations tend to exhibit small and weakly interacting effects in heterozygous loci regardless of how harmful they are in haploids or homozygotes.