What is the underlying cause of type II diabetes? - Are cells protecting themselves against the reactivity of glucose?

Through the last couple of thousands of years, human food sources and availability have changed tremendously. Moving from a diet of hunter-gathers, consisting of nuts, fruits, tubers and meat, to the farmers' diet of vegetables, grains, milk and meat to this and last century, where among other processed foods there is the unlimited availability of refined sugar. This change in diet has been proposed as being the underlying reason for the dramatic increase in the prevalence of type II diabetes that we are observing worldwide in our time. The question is what is the underlying molecular cause of this disease? One possibility proposed here is that type II diabetes might be a consequence of our cells trying to protect themselves from too high intra-cellular concentration of the reactive compound glucose.

The Rim15-endosulfine-PP2ACdc55 signalling module regulates entry into gametogenesis and quiescence via distinct mechanisms in budding yeast.

Quiescence and gametogenesis represent two distinct survival strategies in response to nutrient starvation in budding yeast. Precisely how environmental signals are sensed by yeast cells to trigger quiescence and gametogenesis is not fully understood. A conserved signalling module consisting of Greatwall kinase, Endosulfine and Protein Phosphatase PP2ACdc55 proteins regulates entry into mitosis in Xenopus egg extracts and meiotic maturation in flies. We report here that an analogous signalling module consisting of the serine-threonine kinase Rim15, the Endosulfines Igo1 and Igo2 and the Protein Phosphatase PP2ACdc55, regulates entry into both quiescence and gametogenesis in budding yeast. PP2ACdc55 inhibits entry into gametogenesis and quiescence. Rim15 promotes entry into gametogenesis and quiescence by converting Igo1 into an inhibitor of PP2ACdc55 by phosphorylating at a conserved serine residue. Moreover, we show that the Rim15-Endosulfine-PP2ACdc55 pathway regulates entry into quiescence and gametogenesis by distinct mechanisms. In addition, we show that Igo1 and Igo2 are required for pre-meiotic autophagy but the lack of pre-meiotic autophagy is insufficient to explain the sporulation defect of igo1Δ igo2Δ cells. We propose that the Rim15-Endosulfine-PP2ACdc55 signalling module triggers entry into quiescence and gametogenesis by regulating dephosphorylation of distinct substrates.

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

DNA polymerase α (swi7) and the flap endonuclease Fen1 (rad2) act together in the S-phase alkylation damage response in S. pombe.

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.

Causes and consequences of ribonucleotide incorporation into nuclear DNA.

Intuitively one would not expect that ribonucleotides are incorporated into nuclear DNA beyond their role in priming Okazaki fragments, nor that such incorporation would be functional. However, several recent studies have shown that not only are ribonucleotides present in the nuclear DNA, but that they can be incorporated by at least two different mechanisms: random 'mis'-incorporation of ribonucleotides, which occurs at a surprisingly high frequency; and site-specific incorporation at a stalled fork. Importantly, in the latter case, the ribonucleotides have been shown to have a biological function - acting to initiate a replication-coupled recombination event mediating a cell type change. Traditionally, it has been thought that 'random' ribonucleotide incorporation causes genetic instability, but new evidence suggests there may be a fine balance between mechanisms preventing and incorporating ribonucleotides into genomic DNA. Indeed, genomic ribonucleotides might have diverse roles affecting genetic stability, DNA damage repair, heterochromatin formation, cellular differentiation, and development.

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.

Identification of a novel type of spacer element required for imprinting in fission yeast.

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers.

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.

Random and site-specific replication termination.

Bi-directionality is a common feature observed for genomic replication for all three phylogenetic kingdoms: Eubacteria, Archaea, and Eukaryotes. A consequence of bi-directional replication, where the two replication forks initiated at an origin move away from each other, is that the replication termination will occur at positions away from the origin sequence(s). The replication termination processes are therefore physically and mechanistically dissociated from the replication initiation. The replication machinery is a highly processive complex that in short time copies huge numbers of bases while competing for the DNA substrate with histones, transcription factors, and other DNA-binding proteins. Importantly, the replication machinery generally wins out; meanwhile, when converging forks meet termination occurs, thus preventing over-replication and genetic instability. Very different scenarios for the replication termination processes have been described for the three phylogenetic kingdoms. In eubacterial genomes replication termination is site specific, while in archaea and eukaryotes termination is thought to occur randomly within zones where converging replication forks meet. However, a few site-specific replication barrier elements that mediate replication termination have been described in eukaryotes. This review gives an overview about what is known about replication termination, with a focus on these natural site-specific replication termination sites.

High-resolution mapping of points of site-specific replication stalling.

Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stalled replication fork gives a valuable insight into the causes and mechanisms of replication stalling. The method described here allows mapping of the position of the 3'-end of the nascent leading or lagging strand at the replication fork, stalled at a site-specific barrier. The replicating DNA is purified, digested with restriction enzymes, and enriched by BND-cellulose chromatography. The DNA is separated on a sequencing gel, transferred to a membrane, and hybridised to a strand-specific probe. The data obtained using this method allow determining the position of the 3'-end of the nascent strand at a stalled fork with a one-nucleotide resolution.

Schizosaccharomyces pombe Rtf2 mediates site-specific replication termination by inhibiting replication restart.

Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of "slow-moving" Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.

Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism.

Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.

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.

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.

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

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.

Selective gene expression in multigene families from yeast to mammals.

Exclusive gene expression, where only one member of a gene or gene cassette family is selected for expression, plays an important role in the establishment of cell identity in several biological systems. Here, we compare four such systems: mating-type switching in fission and budding yeast, where cells choose between expressing one of the two different mating-type cassettes, and immunoglobulin and odorant receptor gene expression in mammals, where the number of gene choices is substantially higher. The underlying mechanisms that establish this selective expression pattern in each system differ in almost every detail. In all four systems, once a successful gene activation event has taken place, a feedback mechanism affects the fate of the cell. In the mammalian systems, feedback is mediated by the expressed cell surface receptor to ensure monoallelic gene expression, whereas in the yeasts, the expressed gene cassette at the mating-type locus affects donor choice during the subsequent switching event.

RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching.

Mating-type switching in fission yeast depends on an imprint at the mat1 locus. Previous data showed that the imprint is made in the DNA strand replicated as lagging. We now identify this imprint as an RNase-sensitive modification and suggest that it consists of one or two RNA residues incorporated into the mat1 DNA. Formation of the imprint requires swi1- and swi3-dependent pausing of the replication fork. Interestingly, swi1 and swi3 mutations that abolish pausing do not affect the use of lagging-strand priming site during replication. We show that the pausing of replication and subsequent formation of the imprint occur after the leading-strand replication complex has passed the site of the imprint and after lagging-strand synthesis has initiated at this proximal priming site. We propose a model in which a swi1- and swi3-dependent signal during lagging-strand synthesis leads to pausing of leading-strand replication and the introduction of the imprint.

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 approximately 450 bp containing four repeated 55 bp motifs is essential for function. Secondly, a purine-rich sequence of approximately 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.

A mark in the core: silence no more!

The histone modification repertoire has recently been expanded. Dot1p is a new type of methyltransferase that methylates lysine 79 in the histone H3 core only in its nucleosomal context and has a possible role in marking open chromatin regions.

RTS1-an eukaryotic terminator of replication.

Eukaryotic replication termination generally occurs randomly in the region between two active origins. However, termination, or pausing of the replication forks has been observed at specific loci. Recently, a site-specific terminator of replication named RTS1 was shown to play an important role in mating-type switching in Schizosaccharomyces pombe. Mating-type switching in S. pombe relies on an imprinting event that chemically modifies one strand of the DNA at the mating-type locus mat1. This imprint, that is formed only when mat1 is replicated in a specific direction, marks the DNA for a rearrangement leading to mating-type switching. The RTS1 element ensures that mat1 is replicated in the correct direction for imprinting and initiation of the subsequent mating-type switching event. This is the first replication terminator shown to play a role in cellular differentiation.