22 research outputs found
Inflation on the Brane with Vanishing Gravity
Many existing models of brane inflation suffer from a steep irreducible
gravitational potential between the branes that causes inflation to end too
early. Inspired by the fact that point masses in 2+1 D exert no gravitational
force, we propose a novel unwarped and non-supersymmetric setup for inflation,
consisting of 3-branes in two extra dimensions compactified on a sphere. The
size of the sphere is stabilized by a combination of a bulk cosmological
constant and a magnetic flux. Computing the 4D effective potential between
probe branes in this background, we find a non-zero contribution only from
exchange of level-1 KK modes of the graviton and radion. Identifying antipodal
points on the 2-sphere projects out these modes, eliminating entirely the
troublesome gravitational contribution to the inflationary potential.Comment: 19 pages, 11 figures, JHEP forma
A Dynamic Stochastic Model for DNA Replication Initiation in Early Embryos
Background: Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other studies using Xenopus egg extracts have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation, I(t), markedly increases through S phase to ensure the rapid completion of unreplicated gaps and a narrow distribution of completion times. However, the molecular mechanism that underlies this increase has remained obscure.Methodology/Principal Findings: Using both previous and novel DNA combing data we have confirmed that I(t) increases through S phase but have also established that it progressively decreases before the end of S phase. To explore plausible biochemical scenarios that might explain these features, we have performed comparisons between numerical simulations and DNA combing data. Several simple models were tested: i) recycling of a limiting replication fork component from completed replicons; ii) time-dependent increase in origin efficiency; iii) time-dependent increase in availability of an initially limiting factor, e. g. by nuclear import. None of these potential mechanisms could on its own account for the data. We propose a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins. This novel model quantitatively and robustly accounted for the observed changes in initiation rate and fork density.Conclusions/Significance: This work provides a refined temporal profile of replication initiation rates and a robust, dynamic model that quantitatively explains replication origin usage during early embryonic S phase. These results have significant implications for the organisation of replication origins in higher eukaryotes
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, 4β5 MCM hexamers, and 2 Cdc6. Relative to these subunits, βΌ0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain βΌ70,000 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
Preferential Localization of Human Origins of DNA Replication at the 5β²-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences
Replication of mammalian genomes requires the activation of thousands of
origins which are both spatially and temporally regulated by as yet unknown
mechanisms. At the most fundamental level, our knowledge about the
distribution pattern of origins in each of the chromosomes, among different
cell types, and whether the physiological state of the cells alters this
distribution is at present very limited.We have used standard Ξ»-exonuclease resistant nascent DNA preparations in
the size range of 0.7β1.5 kb obtained from the breast cancer cell line
MCFβ7 hybridized to a custom tiling array containing 50β60 nt
probes evenly distributed among genic and non-genic regions covering about
1% of the human genome. A similar DNA preparation was used for
high-throughput DNA sequencing. Array experiments were also performed with
DNA obtained from BT-474 and H520 cell lines. By determining the sites
showing nascent DNA enrichment, we have localized several thousand origins
of DNA replication. Our major findings are: (a) both array and DNA
sequencing assay methods produced essentially the same origin distribution
profile; (b) origin distribution is largely conserved (>70%) in
all cell lines tested; (c) origins are enriched at the 5β²ends of
expressed genes and at evolutionarily conserved intergenic sequences; and
(d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and
RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely
conserved among different cell types. Also, our work supports recent studies
connecting transcription initiation with replication, and in addition
suggests that evolutionarily conserved intergenic sequences have the
potential to participate in origin selection. Overall, our observations
suggest that replication origin selection is a stochastic process
significantly dependent upon local accessibility to replication factors
Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination
In late mitosis and G1, Mcm2-7 are assembled onto replication origins to βlicenseβ them for initiation. At other times licensing is inhibited by cyclin dependent kinases (CDKs) and geminin, thus ensuring that origins fire only once per cell cycle. We show here that, paradoxically, CDKs are also required to inactivate geminin and activate the licensing system. On exit from metaphase in Xenopus egg extracts, CDK-dependent activation of the Anaphase Promoting Complex (APC/C) results in the polyubiquitination of geminin. This ubiquitination triggers geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed