Mating-type Switching Research Articles

The road not taken: new destinations for yeast mRNAs on the move

The road not taken: new destinations for yeast mRNAs on the move

Histone H3 tail acetylation modulates ATP-dependent remodeling through multiple mechanisms

There is a close relationship between histone acetylation and ATP-dependent chromatin remodeling that is not fully understood. We show that acetylation of histone H3 tails affects SWI/SNF (mating type switching/ sucrose non fermenting) and RSC (remodels structure of chromatin) remodeling in several distinct ways. Acetylation of the histone H3 N-terminal tail facilitated recruitment and nucleosome mobilization by the ATP-dependent chromatin remodelers SWI/SNF and RSC. Tetra-acetylated H3, but not tetra-acetylated H4 tails, increased the affinity of RSC and SWI/SNF for nucleosomes while also changing the subunits of SWI/SNF that interact with the H3 tail. The enhanced recruitment of SWI/SNF due to H3 acetylation is bromodomain dependent, but is not further enhanced by additional bromodomains found in RSC. The combined effect of H3 acetylation and transcription activators is greater than either separately which suggests they act in parallel to recruit SWI/SNF. Besides enhancing recruitment, H3 acetylation increased nucleosome mobilization and H2A/H2B displacement by RSC and SWI/SNF in a bromodomain dependent manner and to a lesser extent enhanced ATP hydrolysis independent of bromodomains. H3 and H4 acetylation did not stimulate disassembly of adjacent nucleosomes in short arrays by SWI/SNF or RSC. These data illustrate how histone acetylation modulates RSC and SWI/SNF function, and provide a mechanistic insight into their collaborative efforts to remodel chromatin.

SCFCdc4 Enables Mating Type Switching in Yeast by Cyclin-Dependent Kinase-Mediated Elimination of the Ash1 Transcriptional Repressor

In the budding yeast Saccharomyces cerevisiae, mother cells switch mating types between a and α forms, whereas daughter cells do not. This developmental asymmetry arises because the expression of the HO endonuclease, which initiates the interconversion of a and α mating type cassettes, is extinguished by the daughter-specific Ash1 transcriptional repressor. When daughters become mothers in the subsequent cell cycle, Ash1 must be eliminated to enable a new developmental state. Here, we report that the ubiquitin ligase SCF(Cdc4) mediates the phosphorylation-dependent elimination of Ash1. The inactivation of SCF(Cdc4) stabilizes Ash1 in vivo, and consistently, Ash1 binds to and is ubiquitinated by SCF(Cdc4) in a phosphorylation-dependent manner in vitro. The mutation of a critical in vivo cyclin-dependent kinase (CDK) phosphorylation site (Thr290) on Ash1 reduces its ubiquitination and rate of degradation in vivo and decreases the frequency of mating type switching. Ash1 associates with active Cdc28 kinase in vivo and is targeted to SCF(Cdc4) in a Cdc28-dependent fashion in vivo and in vitro. Ash1 recognition by Cdc4 appears to be mediated by at least three phosphorylation sites that form two redundant diphosphorylated degrons. The phosphorylation-dependent elimination of Ash1 by the ubiquitin-proteasome system thus underpins developmental asymmetry in budding yeast.

Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS

BackgroundThe S. cerevisiae mating type switch model of double-strand break (DSB) repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR) and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ). We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin.FindingsWe previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1) The site had become less accessible to cleavage by the HO endonuclease; 2) the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3) occupancy of nucleosomes at the site (ChIP for histone H3) was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created. Specifically, the pattern of repair revealed discrete deletions at the DSB that indicated unit losses of nucleosomes (or other protein complexes) before religation, represented by a ladder of PCR products reminiscent of an internucleosomal cleavage that is typically observed during apoptosis. This pattern of cleavage suggested to us that perhaps, Artemis, a protein that is believed to generate the internucleosomal fragments during apoptosis and in DSB repair, was involved in that specific pattern of ends-processing. Preliminary evidence indicates that this may be the case, since knock-down of Artemis with siRNA eliminated the laddering pattern and revealed instead an extensive exonucleolytic processing of the ends before religation.Conclusionse have generated a system in mammalian cells where the absence of positive selection resulted in chromatin remodeling at the target locus that recapitulates many of the features of the mating-type switching system in yeast. Specifically, just as for yeast HML and HMR, the locus had become transcriptionally repressed; accessibility to cleavage by the HO endonuclease was reduced; and processing of the ends was drastically changed. The switch was from high-fidelity religation of the cohesive ends, to a pattern of release of internucleosomal fragments, perhaps in search of micro-homology stretches for ligation. This is consistent with reports that the involvement of ATM, DNA-PK and Artemis in DSB repair is largely focused to heterochromatic regions, and not required for the majority of IR-induced DSB repair foci in euchromatin.

Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination

DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1 and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant displays altered rates of heteroallelic and direct-repeat recombination, sensitivity to DSB-inducing drugs as well as delayed kinetics of mating-type switching with a defect in the DNA synthesis step thus implicating Dpb11 in homologous recombination. We conclude that Dpb11/TopBP1 plays distinct roles in replication, checkpoint response and recombination processes, thereby contributing to chromosomal stability.

Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1

Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.

The yeast mating-type switching mechanism: a memoir.

It has been 33 years since I first presented results of genetic experiments that established the gene transposition model as the mechanism of mating-type switching in the budding yeast Saccharomyces cerevisiae at the Cold Spring Harbor Laboratory (CSHL) Yeast Genetics meeting in August 1977. Over two decades ago the Genetics Perspectives editors solicited a perspective on my participation in the studies that deciphered the mechanism of mating-type switching and revealed the phenomenon of gene silencing in yeast. Although flattered at the time, I thought that preparation of such an article called for a more seasoned researcher who had benefitted from seeing his contributions stand the test of time. Now realizing that our discovery of the transposition of a mutation from the HMα locus into the MAT (mating type) locus has provided the genetic evidence that established the gene transposition model, and having witnessed our conclusions confirmed by subsequent molecular studies, I decided that perhaps this is a good time to recount the chronology of events as they unfolded for me decades ago.

Degradation of the Saccharomyces cerevisiae Mating-Type Regulator α1: Genetic Dissection of Cis-determinants and Trans-acting Pathways

Mating phenotype in the yeast Saccharomyces cerevisiae is a dynamic trait, and efficient transitions between alternate haploid cell types allow the organism to access the advantageous diploid form. Mating identity is determined by cell type-specific transcriptional regulators, but these factors must be rapidly removed upon mating-type switching to allow the master regulators of the alternate state to establish a new gene expression program. Targeted proteolysis by the ubiquitin-proteasome system is a commonly employed strategy to quickly disassemble regulatory networks, and yeast use this approach to evoke efficient switching from the alpha to the a phenotype by ensuring the rapid removal of the alpha2 transcriptional repressor. Transition to the a cell phenotype, however, also requires the inactivation of the alpha1 transcriptional activator, but the mechanism by which this occurs is currently unknown. Here, we report a central role for the ubiquitin-proteasome system in alpha1 inactivation. The alpha1 protein is constitutively short lived and targeted for rapid turnover by multiple ubiquitin-conjugation pathways. Intriguingly, the alpha-domain, a conserved region of unknown function, acts as a degradation signal for a pathway defined by the SUMO-targeted ligase Slx5-Slx8, which has also been implicated in the rapid destruction of alpha2. Our observations suggest coordinate regulation in the turnover of two master regulatory transcription factors ensures a rapid mating-type switch.

Switching the mechanism of mating type switching: a domesticated transposase supplants a domesticated homing endonuclease

Programmed DNA rearrangements are critical for the development of many organisms and, intriguingly, can be catalyzed by domesticated mobile genetic elements. In this issue of Genes & Development, Barsoum and colleagues (pp. 33-44) demonstrate that, in the budding yeast Kluyveromyces lactis, a DNA rearrangement associated with mating type switching requires a domesticated transposase and occurs through a mechanism distinct from that in the related yeast, Saccharomyces cerevisiae. Thus, mechanisms for mating type switching have evolved multiple times, indicating the relative ease with which mobile genetic elements can be captured.

α3, a transposable element that promotes host sexual reproduction

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

Heterothallism inSaccharomyces cerevisiaeisolates from nature: effect ofHOlocus on the mode of reproduction

Understanding the evolution of sex and recombination, key factors in the evolution of life, is a major challenge in biology. Studies of reproduction strategies of natural populations are important to complement the theoretical and experimental models. Fungi with both sexual and asexual life cycles are an interesting system for understanding the evolution of sex. In a study of natural populations of yeast Saccharomyces cerevisiae, we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Mating-type switching is a gene-conversion process initiated by a site-specific endonuclease HO; this process can be followed by mother-daughter mating. Heterothallic yeast can mate with unrelated haploids (amphimixis), or undergo mating between spores from the same tetrad (intratetrad mating, or automixis), but cannot undergo mother-daughter mating as homothallic yeasts can. Sequence analysis of HO gene in a panel of natural S. cerevisiae isolates revealed multiple mutations. Good correspondence was found in the comparison of population structure characterized using 19 microsatellite markers spread over eight chromosomes and the HO sequence. Experiments that tested whether the mating-type switching pathway upstream and downstream of HO is functional, together with the detected HO mutations, strongly suggest that loss of function of HO is the cause of heterothallism. Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating may predominate in natural yeast populations, while mother-daughter mating might not be as significant as was considered.

Mechanisms of polar arrest of a replication fork

A DNA replication terminator sequence blocks an approaching replication fork when the moving replisome approaches from just one direction. The mechanism underlying polar arrest has been debated for years, but recent work has helped to reveal how a replication fork is blocked in Escherichia coli. Early work suggested that asymmetric interaction between terminator protein and terminator DNA contributes to polar fork arrest. A later study demonstrated that if the terminator DNA is partially unwound, the resulting melted DNA could bind tightly to the terminator protein, suggesting a mechanism for polar arrest that involves a locked complex. However, recent evidence suggests that the terminator protein-DNA contacts are not sufficient for polar arrest in vivo. Furthermore, polar arrest of a replication fork still occurs in the absence of a locked complex between the terminator protein and DNA. In E. coli and Bacillus subtilis, the bound terminator protein makes protein-protein contacts with the replication fork helicase, and these contacts are critical in blocking progression of the advancing fork. Thus, we propose that interactions between the replication fork helicase and terminator protein are the primary mechanism for polar fork arrest in bacteria, and that this primary mechanism is modulated by asymmetric contacts between the terminator protein and its cognate DNA sequence. In yeast, terminator sequences are present in rDNA non-transcribed spacers and a region immediately preceding the mating type switch locus Mat1, and the mechanism of polar arrest at these regions is beginning to be elucidated.

Screening a genome-wide S. pombe deletion library identifies novel genes and pathways involved in genome stability maintenance

The maintenance of genome stability is essential for an organism to avoid cell death and cancer. Based on screens for mutant sensitivity against DNA damaging agents a large number of DNA repair and DNA damage checkpoint genes have previously been identified in genetically amenable model organisms. These screens have however not been exhaustive and various genes have been, and remain to be, identified by other means. We therefore screened a genome-wide Schizosaccharomyces pombe deletion library for mutants sensitive against various DNA damaging agents. Screening the library on different concentrations of these genotoxins allowed us to assign a semi-quantitative score to each mutant expressing the degree of sensitivity. We isolated a total of 229 mutants which show sensitivity to one or more of the DNA damaging agents used. This set of mutants was significantly enriched for processes involved in DNA replication, DNA repair, DNA damage checkpoint, response to UV, mating type switching, telomere length maintenance and meiosis, and also for processes involved in the establishment and maintenance of chromatin architecture (notably members of the SAGA complex), transcription (members of the CCR4-Not complex) and microtubule related processes (members of the DASH complex). We also identified 23 sensitive mutants which had previously been classified as “sequence orphan” or as “conserved hypothetical”. Among these, we identified genes showing extensive homology to CtIP, Stra13, Ybp1/Ybp2, Human Fragile X mental retardation interacting protein NUFIP1, and Aprataxin. The identification of these homologues will provide a basis for the further characterisation of the role of these conserved proteins in the genetically amenable model organism S. pombe.

Essential Features of a Non-processive Class V Myosin from Budding Yeast for ASH1 mRNA Transport

A feature of most class V myosins is their ability to move processively on actin. The budding yeast Saccharomyces cerevisiae has a non-processive class V myosin, Myo4p, which is a single-headed but high duty cycle motor. Its cellular role is to asymmetrically transport more than 20 different mRNAs, a widely used strategy to polarize a protein within the cell. The most studied mRNA is ASH1, which is moved by Myo4p to the bud tip to repress mating type switching in the daughter cell. Here we determine the features of Myo4p that are necessary for correct localization of ASH1 mRNA to the daughter cell. This process requires the adapter protein She3p, and the mRNA binding protein She2p, which binds ASH1 at specific localization elements called zip codes. Based on a series of chimeric constructs, we showed that the rod region of Myo4p, but not the globular tail, is essential for correct localization of ASH1 mRNA. The rod thus contains the primary binding site for She3p, consistent with our earlier in vitro studies (Hodges et al., 2008). To test if mRNA localization is more efficient when two motors are coupled together, we compared transport by a constitutive dimer of Myo4p/She3p with a constitutive monomer. Correct ASH1 mRNA localization was achieved equally well with both constructs. This may reflect the fact that many mRNAs and thus many motors are part of the translocation complex. Our results show that the most important feature for correct localization is the retention of coupling between all the members of the complex (Myo4p- She3p-She2p-ASH1 mRNA), which is aided by She3p being a tightly bound subunit of Myo4p.

Msc1 links dynamic Swi6/HP1 binding to cell fate determination

Eukaryotic genomes can be organized into distinct domains of heterochromatin or euchromatin. In the fission yeast Schizosaccharomyces pombe, assembly of heterochromatin at the silent mating-type region is critical for cell fate determination in the form of mating-type switching. Here, we report that the ubiquitin ligase, Msc1, is a critical factor required for proper cell fate determination in S. pombe. In the absence of Msc1, the in vivo mobility of Swi6 at heterochromatic foci is compromised, and centromere heterochromatin becomes hyperenriched with the heterochromatin binding protein Swi6/HP1. However, at the mating-type locus, Swi6 recruitment is defective in the absence of Msc1. Therefore, Msc1 links maintaining dynamic heterochromatin with proper heterochromatin assembly and cell fate determination. These findings have implications for understanding mechanisms of differentiation in other organisms.

Uneven distribution of mating types among genotypes of Candida glabrata isolates from clinical samples.

In order to shed light on its basic biology, we initiated a population genetic analysis of Candida glabrata, an emerging pathogenic yeast with no sexual stage yet recognized. A worldwide collection of clinical strains was subjected to analysis using variable number of tandem repeats (VNTR) at nine loci. The clustering of strains obtained with this method was congruent with that obtained using sequence polymorphism of the NMT1 gene, a locus previously proposed for lineage assignment. Linkage disequilibrium supported the hypothesis of a mainly clonal reproduction. No heterozygous diploid genotype was found. Minimum-spanning tree analysis of VNTR data revealed clonal expansions and associated genotypic diversification. Mating type analysis revealed that 80% of the strains examined are MATa and 20% MATalpha and that the two alleles are not evenly distributed. The MATa genotype dominated within large clonal groups that contained only one or a few MATalpha types. In contrast, two groups were dominated by MATalpha strains. Our data are consistent with rare independent mating type switching events occurring preferentially from type a to alpha, although the alternative possibility of selection favoring type a isolates cannot be excluded.

Second-End Capture in DNA Double-Strand Break Repair Promoted by Brh2 Protein of Ustilago maydis

Brh2 plays a central role in the homologous recombination system of Ustilago maydis, mediating delivery of Rad51 to single-stranded DNA. Here we report that Brh2 can pair the displaced strand of a D loop with a complementary single-stranded DNA to form a duplexed, or double, D loop. The reaction emulates the second-end capture step envisioned in models of DNA double-strand break repair. This second-end capture reaction promoted by Brh2 proceeds efficiently when performed in the presence of Rad51 under conditions that block annealing by Rad52, or when the second single-stranded DNA substrate is replaced by double-stranded DNA. In a coupled reaction that requires extension of the D loop more than 200 nt by DNA synthesis in order to reveal a complementary region, Brh2 was also able to promote second-end capture and thus model a synthesis-dependent strand-annealing mechanism.

Mutants Defective in Rad1-Rad10-Slx4 Exhibit a Unique Pattern of Viability During Mating-Type Switching in Saccharomyces cerevisiae

Efficient repair of DNA double-strand breaks (DSBs) requires the coordination of checkpoint signaling and enzymatic repair functions. To study these processes during gene conversion at a single chromosomal break, we monitored mating-type switching in Saccharomyces cerevisiae strains defective in the Rad1-Rad10-Slx4 complex. Rad1-Rad10 is a structure-specific endonuclease that removes 3' nonhomologous single-stranded ends that are generated during many recombination events. Slx4 is a known target of the DNA damage response that forms a complex with Rad1-Rad10 and is critical for 3'-end processing during repair of DSBs by single-strand annealing. We found that mutants lacking an intact Rad1-Rad10-Slx4 complex displayed RAD9- and MAD2-dependent cell cycle delays and decreased viability during mating-type switching. In particular, these mutants exhibited a unique pattern of dead and switched daughter cells arising from the same DSB-containing cell. Furthermore, we observed that mutations in post-replicative lesion bypass factors (mms2Delta, mph1Delta) resulted in decreased viability during mating-type switching and conferred shorter cell cycle delays in rad1Delta mutants. We conclude that Rad1-Rad10-Slx4 promotes efficient repair during gene conversion events involving a single 3' nonhomologous tail and propose that the rad1Delta and slx4Delta mutant phenotypes result from inefficient repair of a lesion at the MAT locus that is bypassed by replication-mediated repair.

Nuclear localization of Rad52 is pre-requisite for its sumoylation

In Saccharomyces cerevisiae, Rad52 plays major roles in several types of homologous recombination. Here, we found that rad52-K200R mutation greatly reduced sumoylation of Rad52. The rad52-K200R mutant exhibited defects in various types of recombination, such as intrachromosomal recombination and mating-type switching. The K200 residue of Rad52 is part of the nuclear localization signal (NLS), which is important for transport into the nucleus. Indeed, the addition of a SV40 NLS to Rad52-K200R suppressed the sumoylation defect of Rad52-K200R. These findings indicate that nuclear localization of Rad52 is pre-requisite for its sumoylation.

Rad52 sumoylation and its involvement in the efficient induction of homologous recombination

The protein Rad52 is a key player in various types of homologous recombination and is essential to maintenance of genomic integrity. Although evidence indicates that Rad52 is modified by SUMO, the physiological relevance of this sumoylation remains unclear. Here, we identify the conditions under which Rad52 sumoylation is induced, and clarify the role of this modification in homologous recombination. Oligomerization of Rad52 was a prerequisite for sumoylation, and the modification occurred in the cell proceeding S phase being exposed to the DNA-damaging agent methyl methanesulfonate (MMS). Following exposure to MMS, sumoylated Rad52 accumulated in rad51 cells, but not in the recombination-related gene mutants, rad54, rad55, rad59, sgs1, or srs2. The accumulation of sumoylated Rad52 was suppressed in rad51 cells expressing Rad51-K191R, an ATPase-defective protein presumed to be recruited to ssDNA. Although the sumoylation defective mutant rad52- 3KR (K10R/K11R/K220R) showed no defect in mating-type switching, which did not lead to Rad52 sumoylation in wild-type cells, the mutant did demonstrate a partial defect in MMS-induced interchromosomal homologous recombination.