Resolution of recombination intermediates generated during yeast mating type switching

Interchromosomal gene conversion between alleles has been shown in yeast frequently to be associated with the recombination of flanking genetic markers. Although this also holds true for gene conversion between two alleles of the yeast mating-type (MAT) locus, initiated by the homothallic switching system, we find no evidence that crossing-over ever accompanies gene conversion between the non-allelic HMR and MAT genes when initiated by this same homothallic switching system.     

Double-strand breaks at an initiation site for meiotic gene conversion

It has been proposed that the initiation of meiotic recombination involves either single-strand or double-strand breaks in DNA. It is difficult to distinguish between these on the basis of genetic evidence because they give rise to similar predictions. All models invoke initiation at specific sites to explain polarity, which is a gradient in gene conversion frequency from one end of a gene to the other. In the accompanying paper we describe the localization of an initiation site for gene conversion to the promoter region of the ARG4 gene of the yeast Saccharomyces cerevisiae. Here, we show that a double-strand break appears at the ARG4 recombination initiation site at the time of recombination, and that the broken DNA molecules end in long single-stranded tails.     

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.     

Microconversion between murine H-2 genes integrated into yeast

Patchwork homology observed between divergent members of polymorphic multigene families is thought to reflect evolution by short-tract gene conversion (nonreciprocal recombination), although this mechanism cannot usually be confirmed in higher organisms. In contrast to meiotic conversions observed in laboratory yeast strains, apparent conversions between polymorphic sequences, such as the class I loci of the major histocompatibility complex (MHC), are short and do not seem to be associated with reciprocal recombination (crossover, exchanges). We have now integrated two nonallelic murine class I genes into yeast to characterize their meiotic recombination. We found no crossovers between the MHC genes, but short-tract 'microconversions' of 1-215 base-pairs were observed in about 6% of all meioses. Strikingly, one of these events was accompanied by a single base-pair mutation. These results underscore both the importance of meiotic gene conversion and sequence heterology in determining conversion patterns between divergent genes.     

High-resolution mapping of meiotic crossovers and non-crossovers in yeast

Meiotic recombination has a central role in the evolution of sexually reproducing organisms. The two recombination outcomes, crossover and non-crossover, increase genetic diversity, but have the potential to homogenize alleles by gene conversion. Whereas crossover rates vary considerably across the genome, non-crossovers and gene conversions have only been identified in a handful of loci. To examine recombination genome wide and at high spatial resolution, we generated maps of crossovers, crossover-associated gene conversion and non-crossover gene conversion using dense genetic marker data collected from all four products of fifty-six yeast (Saccharomyces cerevisiae) meioses. Our maps reveal differences in the distributions of crossovers and non-crossovers, showing more regions where either crossovers or non-crossovers are favoured than expected by chance. Furthermore, we detect evidence for interference between crossovers and non-crossovers, a phenomenon previously only known to occur between crossovers. Up to 1% of the genome of each meiotic product is subject to gene conversion in a single meiosis, with detectable bias towards GC nucleotides. To our knowledge the maps represent the first high-resolution, genome-wide characterization of the multiple outcomes of recombination in any organism. In addition, because non-crossover hotspots create holes of reduced linkage within haplotype blocks, our results stress the need to incorporate non-crossovers into genetic linkage analysis.     

Template switching during break-induced replication

DNA double-strand breaks (DSBs) are potentially lethal lesions that arise spontaneously during normal cellular metabolism, as a consequence of environmental genotoxins or radiation, or during programmed recombination processes. Repair of DSBs by homologous recombination generally occurs by gene conversion resulting from transfer of information from an intact donor duplex to both ends of the break site of the broken chromosome. In mitotic cells, gene conversion is rarely associated with reciprocal exchange and thus limits loss of heterozygosity for markers downstream of the site of repair and restricts potentially deleterious chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). BIR from one of the two ends of a DSB would result in loss of heterozygosity, suggesting that BIR is suppressed when DSBs have two ends so that repair occurs by the more conservative gene conversion mechanism. Here we show that BIR can occur by several rounds of strand invasion, DNA synthesis and dissociation. We further show that chromosome rearrangements can occur during BIR if dissociation and reinvasion occur within dispersed repeated sequences. This dynamic process could function to promote gene conversion by capture of the displaced invading strand at two-ended DSBs to prevent BIR.     

Break-induced replication and telomerase-independent telomere maintenance require Pol32

Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template. BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telomeres in cells that lack telomerase in pathways known as 'alternative lengthening of telomeres' (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Polalpha-primase complex as well as Poldelta to initiate new DNA synthesis. Polepsilon is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Poldelta subunit Pol32 primarily through its interaction with another Poldelta subunit, Pol31. HO-induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51-dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination-dependent DNA replication. Thus Pol32, dispensable for replication and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA replication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified.     

染色体位置对酵母SUC2基因表达的影响

通过ＦＬＰ－ＦＲＴ位点特异性ＤＮＡ切离和ＤＮＡ定点整合技术,将酵母蔗糖酶基因整合到酿酒酵母染色体不同位置上,测定了ＳＵＣ２基因表达情况,从而对酵母染色体位置效应进行了初步研究。     

Isolation, characterization and functional analysis of a cdc48 homologue from tobacco BY-2 cells

The fission yeast Schizosaccharomyces pombe was used to identify genes from tobacco BY-2 cells that may play roles in cell cycle regulation. A cDNA encoding a protein homologous to the yeast CDC48 was isolated and the gene was designated as NtCDC48. The cDNA contains an open reading frame coding for a predicted protein of 808 amino acids which comprises of two typical ATPase modules (aa 245?374 and aa 518?646). Overexpression of NtCDC48 in tobacco BY-2 cells led to an increase in the mitotic index as well as to the formation of diffused mitotic spindles. NtCDC48-GFP fusion proteins are distributed ubiquitously through G1 to M phases, yet their subcellular localization varied regularly along with the cell cycle progression. These results indicate that NtCDC48 may play an important role in the regulation of cell cycle in BY-2 cells.     

Isolation, characterization and functional analysis of a cdc48 homologue from tobacco BY-2 cells

The fission yeast Schizosaccharomyces pombe was used to identify genes from tobacco BY-2 cells that may play roles in cell cycle regulation. A cDNA encoding a protein homologous to the yeast CDC48 was isolated and the gene was designated as NtCDC48 . The cDNA contains an open reading frame coding for a predicted protein of 808 amino acids which comprises of two typical ATPase modules (aa 245-374 and aa 518-646) . Overexpression of NtCDC48 in tobacco BY-2 cells led to an increase in the mitotic index as well as to the formation of diffused mitotic spindles. NtCDC48-GFP fusion proteins are distributed ubiquitously through Gl to M phases, yet their subcellular localization varied regularly along with the cell cycle progression. These results indicate that NtCDC48 may play an important role in the regulation of cell cycle in BY-2 cells.     

同源重组法敲除酵母snoRNA基因的初步探讨

以3个粟酒裂殖酵母核仁小RNA为对象,通过同源重组法构建相应的基因缺失株,并对影响同源重组效率的因素进行分析.结果显示:载体骨架的切除可以显著提高同源重组效率;增加两侧同源片段的长度也能提高同源重组效率;另外,利用粟酒裂殖酵母野生型单倍体菌株进行snoRNA 基因敲除是可行的.     

Telomere-telomere recombination provides an express pathway for telomere acquisition

DNA termini from Tetrahymena and Oxytricha, which bear C4A2 and C4A4 repeats respectively, can support telomere formation in Saccharomyces cerevisiae by serving as substrates for the addition of yeast telomeric C1-3A repeats. Previously, we showed that linear plasmids with 108 base pairs of C4A4 DNA (YLp108CA) efficiently acquired telomeres, whereas plasmids containing 28-64 base pairs of C4A4 DNA also promoted telomere formation, but with reduced efficiency. Although many of the C4A4 termini on these plasmids underwent recombination with a C4A2 terminus, the mechanism of telomere-telomere recombination was not established. We now report the sequence of the C4A4 ends from the linear plasmids. The results provide strong evidence for a novel recombination process involving a gene conversion event that requires little homology, occurs at or near the boundary of telomeric and nontelomeric DNA, and resembles the recombination process involved in bacteriophage T4 DNA replication.     

Genome-wide genetic analysis of polyploidy in yeast

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.     

A meiotic gene conversion gradient opposite to the direction of transcription.

Genetic recombination involves classical crossing-over and gene conversion (aberrant segregation). In fungi that produce an ascus containing four spores, a gene conversion event is manifested as 3:1 or 1:3 (or more rarely 4:0 or 0:4) segregations, in contrast to the normal mendelian 2:2 segregation. Polarity is one of the properties of gene conversion; in almost all cases the frequency of conversion exhibits a gradient across the gene monitored. The frequency of conversion is usually independent of the specific allele used as a marker, but dependent on its location. An interpretation of conversion polarity is that it is caused by the existence of specific initiation sites for meiotic recombination, located at the high end of the polarity gradient. Here we show that the polarity gradient for the HIS2 gene of Saccharomyces cerevisiae is high at the 3' end of the gene, implying that the promoter of HIS2 is not the initiation site.     

Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast.

Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle. The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene. Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S. pombe, one of which is associated with the mitotic spindle poles. Both populations colocalize with the product of the cdc2 gene (p34cdc2). Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2. These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.     

Heterochromatin links to centromeric protection by recruiting shugoshin

The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6 cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.     

Renaturation of DNA catalysed by yeast DNA repair and recombination protein RAD10.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of ultraviolet-damaged DNA, and it functions in mitotic recombination. RAD10 has homology to the human excision repair gene ERCC-1. Here we describe the purification of the protein encoded by RAD10 and show that it is a DNA-binding protein with a strong preference for single-stranded DNA. We also show that RAD10 promotes the renaturation of complementary DNA strands.     

Wee1(+)-like gene in human cells.

The wee1+ gene is a mitotic inhibitor controlling the G2 to M transition of the fission yeast Schizosaccharomyces pombe and encodes a protein kinase with both serine- and tyrosine-phosphorylating activities. We have cloned a human gene (WEE1Hu) similar to wee1+ by transcomplementation of a yeast mutant. WEE1Hu encodes a protein homologous to the S. pombe wee1+ and mik1+ (a functionally redundant sibling of wee1+) kinases and effectively rescues a wee1 mutation. We report here that overexpression of WEE1Hu in fission yeast generates very elongated cells as a result of inhibition of the G2-M transition in the cell cycle. In addition, we detected a 3-kilobase-long WEE1Hu messenger RNA in all the human cell lines we examined. We conclude that a wee1(+)-like gene exists and is expressed in human cells.     

Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene

The structure equivalent to higher eukaryotic centrosomes in fission yeast, the nuclear membrane-bound spindle pole body, is inactive during interphase. On transition from G2 to M phase of the cell cycle, the spindle pole body duplicates; the daughter pole bodies seed microtubules which interdigitate to form a short spindle that elongates to span the nucleus at metaphase. We have identified two loci which, when mutated, block spindle formation. The predicted product of one of these genes, cut7+, contains an amino-terminal domain similar to the kinesin heavy chain head domain, indicating that the cut7+ product could be a spindle motor. The cut7+ gene resembles the Aspergillus nidulans putative spindle motor gene bimC, both in terms of its organization with a homologous amino-terminal head and no obvious heptad repeats and in the morphology of the mutant phenotype. But we find no similarity between the carboxy termini of these genes, suggested that either the cut7+ gene represents a new class of kinesin genes and that fission yeast may in addition contain a bimC homologue, or that the carboxy termini of these mitotic kinesins are not evolutionarily conserved and that the cut7+ gene belongs to a subgroup of bimC-related kinesins.