A Proteomic and Genomic Investigation Into Replication Fork Dynamics in Saccharomyces Cerevisiae

In eukaryotic organisms, each chromosome must be precisely replicated every time a cell divides so that the genetic material can be passed on to the cell’s progeny. The work presented here is an in-depth investigation into the dynamics of the proteins that associate with progressing replication forks in yeast. A focused proteomics approach is employed to specifically identify interactions between the replication fork-coupled GINS complex and other components of the replication machinery. The scope of this technique is extended by applying it to cells that have been synchronized within the cell cycle – revealing the cell cycle dependent interactions of the GINS complex. The results show that GINS is a stable complex throughout the cell cycle, and interacts with components of the replicative helicase and chromatin during S –phase. Previous studies have led to a picture wherein the replication of DNA progresses at variable rates over different parts of the budding yeast genome. It is widely held that the dynamics of replication fork progression are strongly affected by local chromatin structure/architecture, and by interaction with machineries controlling transcription, repair and epigenetic maintenance. Here we adopted a complementary approach to those previously applied for assaying replication dynamics wherein we used whole genome time-resolved ChIP-chip analysis of three integral members of the replication fork – the GINS complex, Polymerase !, and Polymerase . Surprisingly, our data demonstrate that these proteins progress at highly uniform rates regardless of genomic location, revealing that replication fork dynamics in yeast is simpler and more uniform than previously envisaged. In addition, we demonstrate how the synergistic use of experiment and modeling leads to novel biological insights. In particular, a parsimonious model allowed us to accurately simulate fork movement throughout the genome and also revealed a subtle phenomenon, which we interpret as arising from low frequency fork arrest. Taken together, these experiments suggest that the progressing replication forks take precedence in the genome, and that chromatin state does not have as significant affect on the rate of fork progression, as was previously believed

Structure-function mapping of a heptameric module in the nuclear pore complex.

The nuclear pore complex (NPC) is a multiprotein assembly that serves as the sole mediator of nucleocytoplasmic exchange in eukaryotic cells. In this paper, we use an integrative approach to determine the structure of an essential component of the yeast NPC, the ~600-kD heptameric Nup84 complex, to a precision of ~1.5 nm. The configuration of the subunit structures was determined by satisfaction of spatial restraints derived from a diverse set of negative-stain electron microscopy and protein domain-mapping data. Phenotypic data were mapped onto the complex, allowing us to identify regions that stabilize the NPC's interaction with the nuclear envelope membrane and connect the complex to the rest of the NPC. Our data allow us to suggest how the Nup84 complex is assembled into the NPC and propose a scenario for the evolution of the Nup84 complex through a series of gene duplication and loss events. This work demonstrates that integrative approaches based on low-resolution data of sufficient quality can generate functionally informative structures at intermediate resolution

A Biochemically Active MCM-like Helicase in Bacillus Cereus

The mini-chromosome maintenance (MCM) proteins serve as the replicative helicases in archaea and eukaryotes. Interestingly, an MCM homolog was identified, by BLAST analysis, within a phage integrated in the bacterium Bacillus cereus (Bc). BcMCM is only related to the AAA region of MCM-helicases; the typical amino-terminus is missing and is replaced by a segment with weak homology to primases. We show that BcMCM displays 3'-->5' helicase and ssDNA-stimulated ATPase activity, properties that arise from its conserved AAA domain. Isolated BcMCM is a monomer in solution but likely forms the functional oligomer in vivo. We found that the BcMCM amino-terminus can bind ssDNA and harbors a zinc atom, both hallmarks of the typical MCM amino-terminus. No BcMCM-catalyzed primase activity could be detected. We propose that the divergent amino-terminus of BcMCM is a paralog of the corresponding region of MCM-helicases. A divergent amino terminus makes BcMCM a useful model for typical MCM-helicases since it accomplishes the same function using an apparently unrelated structure.Molecular and Cellular Biolog

GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome

Time-resolved ChIP-chip can be utilized to monitor the genome-wide dynamics of the GINS complex, yielding quantitative information on replication fork movement.Replication forks progress at remarkably uniform rates across the genome, regardless of location.GINS progression appears to be arrested, albeit with very low frequency, at sites of highly transcribed genes.Comparison of simulation with data leads to novel biological insights regarding the dynamics of replication fork progressio

Stochastic association of neighboring replicons creates replication factories in budding yeast

Peer reviewedPublisher PD

The dynamics of genome replication using deep sequencing

Peer reviewedPublisher PD

OriDB, the DNA replication origin database updated and extended

OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted replication origin sites. The original database collated and curated Saccharomyces cerevisiae origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated origin mapping studies, and to include the collation of replication origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/

Mutation Rates across Budding Yeast Chromosome VI Are Correlated with Replication Timing

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation

Do replication forks control late origin firing in Saccharomyces cerevisiae?

Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution. The single cell I(t) and the population-averaged I(t) extracted from DNA combing and replication timing profiles are similar, indicating a genome scale invariance of the replication process, and excluding cell-to-cell variability in replication time as an explanation for the shape of I(t). The single cell I(t) correlates with fork density in wild-type cells, which is specifically loosened in late S phase in the clb5Δ mutant. A previously proposed numerical model that reproduces the wild-type I(t) profile, could also describe the clb5Δ mutant I(t) once modified to incorporate the decline in CDK activity and the looser dependency of initiation on fork density in the absence of Clb5p. Overall, these results suggest that the replication forks emanating from early fired origins facilitate origin firing in later-replicating regions

Nuclear DNA Replication in Trypanosomatids:There Are No Easy Methods for Solving Difficult Problems

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens