Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation

Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

<p>Abstract</p> <p>Background</p> <p>Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.</p> <p>Findings</p> <p>Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, <it>Schizosaccharomyces pombe</it>. To preserve <it>in vivo </it>molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.</p> <p>Conclusions</p> <p>We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.</p

Laboratories, laws, and the career of a commodity

Unlike most foods, milk is produced fresh at least twice every day, thus recreating, over 700 times a year, a commodity ‘designed’ by the combination of nature, commerce, and law. The paper is a study of the ontogenesis of this commodity in Britain since 1800, stressing the emergence of two new objectivities: dairy science and the law on adulteration. In the words of Christopher Hamlin, what mattered was the “manufacture of certainty, however flimsy that certainty might later be shown to be.'' This was achieved by the collection of samples, the generation of facts by the deployment of the laboratory technologies of physics and chemistry, and a semimonopoly over the truth-power of dairy science that was gradually built up by the large commercial companies. A foundation of state-sponsored regulation provided an official legitimation of compositional standards that suited the interests of capital but ignored ‘natural’ variations in quality and often pilloried innocent producers. The public eventually became accustomed to the regulated quality of the milk in its ‘pinta’ and assumed it to be natural. Even the standardization of composition since 1993 has caused very little disquiet among the consuming public, although milk is now a fully constructed commodity like any other dairy product. Mechanical modernity has at last triumphed over a century of ‘milk as it came from the cow’

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

Cdc6 Chromatin Affinity Is Unaffected by Serine-54 Phosphorylation, S-Phase Progression, and Overexpression of Cyclin A

Ectopically expressed Cdc6 is translocated from the nucleus during S phase in a cyclin A-Cdk2-dependent process, suggesting that reinitiation of DNA replication is prevented by removal of phosphorylated Cdc6 from chromatin after origin firing. However, whether endogenous Cdc6 translocates during S phase remains controversial. To resolve the questions regarding regulation of endogenous Cdc6, we cloned the cDNA encoding the Chinese hamster Cdc6 homolog and specifically focused on analyzing the localizations and chromatin affinities of endogenous and exogenous proteins during S phase and following overexpression of cyclin A. In agreement with other reports, ectopically expressed Cdc6 translocates from the nucleus during S phase and in response to overexpressed cyclin A. In contrast, using a combination of biochemical and immunohistochemical assays, we show convincingly that endogenous Cdc6 remains nuclear and chromatin bound throughout the entire S period, while Mcm5 loses chromatin affinity during S phase. Overexpression of cyclin A is unable to alter the nuclear localization of Cdc6. Furthermore, using a phosphospecific antibody we show that phosphoserine-54 Cdc6 maintains a high affinity for chromatin during the S period. Considering recent in vitro studies, these data are consistent with a proposed model in which Cdc6 is serine-54 phosphorylated during S phase and functions as a chromatin-bound signal that prevents reformation of prereplication complexes

Specific signals at the 3′ end of the DHFR gene define one boundary of the downstream origin of replication

The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a 55-kb zone of potential initiation sites lying between the convergently transcribed DHFR and 2BE2121 genes. Two subregions within this zone (ori-β/ori-β′ and ori-γ) are preferred. In the DHFR-deficient variant, DR8, which has deleted a 14-kb sequence straddling the 3′ end of the DHFR gene, early-firing origin activity in the downstream ori-β/ori-β′ and ori-γ regions is completely suppressed. We show that the critical deleted sequences reside within a 168-bp segment encompassing the intron 5/exon 6 boundary, exon 6, 54 bp of the 3′ untranslated region (UTR), but not the three natural polyA sites. In wild-type cells, this sequence efficiently arrests transcription in a region a few kilobases downstream, which coincides with the 5′ boundary of the replication initiation zone. In DR8, DHFR-specific transcripts efficiently use an alternative sixth exon (6c) and polyA signals near the middle of the former intergenic region to process primary transcripts. However, transcription proceeds to a position almost 35 kb downstream from these signals, and replication initiation can only be detected beyond this point. When the wild-type 168-bp 3′ element is inserted into DR8 at the same position as alternative exon 6c, transcription is arrested efficiently and initiations occur almost immediately downstream. Thus, the normal 3′ end of the DHFR gene constitutes a boundary element not only for the gene but also for the local origin of replication