Jacob Z. Dalgaard

Comparative Functional Genomics of the Fission Yeasts

A combined analysis of genome sequence, structure, and expression gives insights into fission yeast biology. The fission yeast clade—comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus—occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions

Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5–Smc6 complexes. The Smc5–Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5–Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5–Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.

Orientation of DNA replication establishes mating-type switching pattern in S. pombe

The fission yeast Schizosaccharomyces pombe normally has haploid cells of two mating types, which differ at the chromosomal locus mat1. After two consecutive asymmetric cell divisions, only one in four ‘grand-daughter’ cells undergoes a ‘mating-type switch’, in which genetic information is transferred to mat1 from the mat2-P or mat3-M donor loci. This switching pattern probably results from an imprinting event at mat1 that marks one sister chromatid in a strand-specific manner, and is related to a site-specific, double-stranded DNA break at mat1,. Here we show that the genetic imprint is a strand-specific, alkali-labile DNA modification at mat1. The DNA break is an artefact, created from theimprint during DNA purification. We also propose and test themodel that mat1 is preferentially replicated by a centromere-distal origin(s), so that the strand-specific imprint occurs only during lagging-strand synthesis. Altering the origin of replication, by inverting mat1 or introducing an origin of replication, affects the imprinting and switching efficiencies in predicted ways. Two-dimensional gel analysis confirmed that mat1 is preferentially replicated by a centromere-distal origin(s). Thus, the DNA replication machinery may confer different developmental potential to sister cells.

Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage

ABSTRACT The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.

The wild‐type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides

The imprint at the mat1 locus of Schizosaccharomyces pombe acts to initiate the replication‐coupled recombination event that underlies mating‐type switching. However, the nature of the imprint has been an area of dispute. Two alternative models have been proposed: one stated that the imprint is a nick in the DNA, whereas our data suggested that it consists of one or two ribonucleotides incorporated into the otherwise intact DNA duplex. Here, we verify key predictions of the RNA model by characterization of wild‐type genomic DNA purified under conditions known to hydrolyse DNA–RNA–DNA hybrid strands. First, we observe one‐nucleotide gap at the hydrolysed DNA, as expected from the presence of two ribonucleotides. Second, using a novel assay based on ligation‐mediated PCR, a 3′‐terminal ribonucleotide is detected at the hydrolysed imprint. Our observations allow the unification of available data sets characterizing the wild‐type imprint.

Does S. pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching?

Typically cell division is envisaged to be symmetrical, with both daughter cells being identical. However, during development and cellular differentiation, asymmetrical cell divisions have a crucial role. In this article, we describe a model of how Schizosaccharomyces pombe exploits the intrinsic asymmetry of DNA replication machinery--the difference between the replication of the leading strand and the lagging strand--to establish an asymmetrical mating-type switching pattern. This is the first system where the direction of DNA replication is involved in the formation of differentiated chromosomes. The discovery raises the possibility that DNA replication might be more generally involved in the establishment of asymmetric cellular differentiation.

The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability

Bidirectionally moving DNA replication forks merge at termination sites composed of accidental or programmed DNA-protein barriers. If merging fails, then regions of unreplicated DNA can result in the breakage of DNA during mitosis, which in turn can give rise to genome instability. Despite its importance, little is known about the mechanisms that promote the final stages of fork merging in eukaryotes. Here we show that the Pif1 family DNA helicase Pfh1 plays a dual role in promoting replication fork termination. First, it facilitates replication past DNA-protein barriers, and second, it promotes the merging of replication forks. A failure of these processes in Pfh1-deficient cells results in aberrant chromosome segregation and heightened genome instability.

Complex mechanism of site-specific DNA replication termination in fission yeast

A site‐specific replication terminator, RTS1, is present at the Schizosaccharomyces pombe mating‐type locus mat1. RTS1 regulates the direction of replication at mat1, optimizing mating‐type switching that occurs as a replication‐coupled recombination event. Here we show that RTS1 contains two cis‐acting sequences that cooperate for efficient replication termination. First, a sequence of ∼450 bp containing four repeated 55 bp motifs is essential for function. Secondly, a purine‐rich sequence of ∼60 bp without intrinsic activity, located proximal to the repeats, acts cooperatively to increase barrier activity 4‐fold. Our data suggest that the trans‐acting factors rtf1p and rtf2p act through the repeated motifs and the purine‐rich element, respectively. Thus, efficient site‐specific replication termination at RTS1 occurs by a complex mechanism involving several cis‐acting sequences and trans‐acting factors. Interestingly, RTS1 displays similarities to mammalian rDNA replication barriers.

Rtf1-Mediated Eukaryotic Site-Specific Replication Termination

The molecular mechanisms mediating eukaryotic replication termination and pausing remain largely unknown. Here we present the molecular characterization of Rtf1 that mediates site-specific replication termination at the polar Schizosaccharomyces pombe barrier RTS1. We show that Rtf1 possesses two chimeric myb/SANT domains: one is able to interact with the repeated motifs encoded by the RTS1 element as well as the elements enhancer region, while the other shows only a weak DNA binding activity. In addition we show that the C-terminal tail of Rtf1 mediates self-interaction, and deletion of this tail has a dominant phenotype. Finally, we identify a point mutation in Rtf1 domain I that converts the RTS1 element into a replication barrier of the opposite polarity. Together our data establish that multiple protein DNA and protein–protein interactions between Rtf1 molecules and both the repeated motifs and the enhancer region of RTS1 are required for site-specific termination at the RTS1 element.

The many facets of the Tim-Tipin protein families' roles in chromosome biology.

Failures in DNA replication are a potent force for driving genome instability. The proteins which form the replisome, the DNA replication machinery, play a fundamental role in preventing replicative catastrophes. The Tim (TIMELESS/TIMEOUT) and Tipin proteins are two conserved replisome associated proteins which have functions in preventing replication fork collapse and replicative checkpoint signalling in response to factors which slow the progression of the replisome. Intriguingly, TIMELESS family members have been implicated in the regulation of the biological clock, giving a tantalising pointer to a possible link between DNA replication and circadian rhythm control. Here we report on our current understanding of the many facets of these protein families in maintaining genome stability and replication checkpoint control.