Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences

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

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms

Indicator Geostatistical Analysis of Sand Interconnections Within a Till

At the Fernald Environmental Management Project (FEMP) site in southwestern Ohio, sand and gravel lenses within till provide potential pathways for the flow of contamination into the underlying aquifer. These high-permeability lenses account for 22% of the volume of the till, have a complex arrangement, and are smaller in scale than the site. Even the relatively dense subsurface sampling program at the FEMP site is not adequate to determine facies interconnections with certainty. To delineate probable facies boundaries, a binary indicator random variable was used to represent the presence of high or low-permeability sediment. The 800 available lithologic logs that penetrate the till were coded with the binary system at 2 ft (0.6 m) intervals yielding 15,829 observations. These data were used to compute the declustered mean of indicator values in horizontal intervals, giving an estimate of the proportion of high-permeability sediment in each vertical zone. The areal correlation in specific zones was examined through indicator variograms, which had pronounced anisotropy. Three-dimensional indicator point kriging was used to produce maps of the probability of existence of high-permeability sediment. These maps were used in a preliminary analysis of sand body interconnectedness. Results indicate that portions of sand bodies are interconnected through the entire interval studied and that the areal extent of vertical interconnection is up to 1000 ft

Indicator Geostatistical Analysis of Sand Interconnections Within a Till

At the Fernald Environmental Management Project (FEMP) site in southwestern Ohio, sand and gravel lenses within till provide potential pathways for the flow of contamination into the underlying aquifer. These high-permeability lenses account for 22% of the volume of the till, have a complex arrangement, and are smaller in scale than the site. Even the relatively dense subsurface sampling program at the FEMP site is not adequate to determine facies interconnections with certainty. To delineate probable facies boundaries, a binary indicator random variable was used to represent the presence of high or low-permeability sediment. The 800 available lithologic logs that penetrate the till were coded with the binary system at 2 ft (0.6 m) intervals yielding 15,829 observations. These data were used to compute the declustered mean of indicator values in horizontal intervals, giving an estimate of the proportion of high-permeability sediment in each vertical zone. The areal correlation in specific zones was examined through indicator variograms, which had pronounced anisotropy. Three-dimensional indicator point kriging was used to produce maps of the probability of existence of high-permeability sediment. These maps were used in a preliminary analysis of sand body interconnectedness. Results indicate that portions of sand bodies are interconnected through the entire interval studied and that the areal extent of vertical interconnection is up to 1000 ft

Explaining the Thinness of Fresh Water Lenses in the Pleistocene Carbonate Aquifer on Andros Island, Bahamas

Two hypotheses are explored to explain the thinness of the fresh water lens on Andros Island, Bahamas. The lens is an order of magnitude thinner than predicted by the Ghyben-Herzberg theory. One hypothesis previously posed in the literature is that the base of the lens is governed by the contact between the Lucayan Formation and the pre-Lucayan limestones. An alternate hypothesis, posed here, is that thinning is caused by the hydraulic influence of low-permeability paleosols in the Lucayan Formation. These hypotheses were explored, along with the influence of recharge and other factors, using a numerical model for variable-density flow and salt transport. In the layered system of grainstones/packstones and paleosols within the Lucayan Formation itself, the velocity vectors are nearly horizontal in the grainstones/packstones, and they are nearly vertical in the paleosols. These strata above the pre-Lucayan, all lower than it in permeability, draw the base of the lens upward to a position well above the contact by significantly reducing pressure within and below the strata relative to hydrostatic pressure. It is the loss of pressure across the paleosols that dramatically thins the fresh water lens, and thus the predominant hydraulic control on lens thickness arises from the existence of paleosols

Challenging Geostatistical Methods to Represent Heterogeneity in CO2 Reservoirs Under Residual Trapping

Geostatistical methods based on two-point spatial-bivariate statistics have been used to model heterogene­ity within computational studies of the dispersion of con­taminants in groundwater reservoirs and the trapping ofCO2 in geosequestration reservoirs. The ability of these methods to represent fluvial architecture, commonly oc­curring in such reservoirs, has been questioned. We challenged a widely used two-point spatial-bivariate sta­tistical method to represent fluvial heterogeneity in the context of representing how reservoir heterogeneity af­fects residual trapping of CO2 injected for geosequestra­tion. A more rigorous model for fluvial architecture was used as the benchmark in these studies. Both the geo-statistically generated model and the benchmark model were interrogated, and metrics for the connectivity of high-permeability preferential flow pathways were quan­tified. Computational simulations of CO2 injection were performed, and metrics for CO2 dynamics and trapping were quantified. All metrics were similar between the two models. The percentage of high-permeability cells in spanning connected clusters (percolating clusters) was similar because percolation is strongly dependent upon proportions, and the same proportion of higher per­meability cross-strata was specified in generating both models. The CO2 plume dynamics and residual trapping metrics were similar because they are largely controlled by the occurrence of percolating clusters. The bench­mark model represented more features of the fluvial ar­chitecture and, depending on context, representing those features may be quite important, but the simpler geosta­tistical model was able to adequately represent fluvialreservoir architecture within the context and within the scope of the parameters represented here