Genome-wide estimation of firing efficiencies of origins of DNA replication from time-course copy number variation data

<p>Abstract</p> <p>Background</p> <p>DNA replication is a fundamental biological process during S phase of cell division. It is initiated from several hundreds of origins along whole chromosome with different firing efficiencies (or frequency of usage). Direct measurement of origin firing efficiency by techniques such as DNA combing are time-consuming and lack the ability to measure all origins. Recent genome-wide study of DNA replication approximated origin firing efficiency by indirectly measuring other quantities related to replication. However, these approximation methods do not reflect properties of origin firing and may lead to inappropriate estimations.</p> <p>Results</p> <p>In this paper, we develop a probabilistic model - Spanned Firing Time Model (SFTM) to characterize DNA replication process. The proposed model reflects current understandings about DNA replication. Origins in an individual cell may initiate replication randomly within a time window, but the population average exhibits a temporal program with some origins replicated early and the others late. By estimating DNA origin firing time and fork moving velocity from genome-wide time-course S-phase copy number variation data, we could estimate firing efficiency of all origins. The estimated firing efficiency is correlated well with the previous studies in fission and budding yeasts.</p> <p>Conclusions</p> <p>The new probabilistic model enables sensitive identification of origins as well as genome-wide estimation of origin firing efficiency. We have successfully estimated firing efficiencies of all origins in S.cerevisiae, S.pombe and human chromosomes 21 and 22.</p

High-Resolution Replication Profiles Define the Stochastic Nature of Genome Replication Initiation and Termination

Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.Peer reviewedPublisher PD

Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics

The involvement of single-stranded DNA, replication protein A, and the DNA double-strand break dose in the damage checkpoint of Saccharomyces cerevisiae.

In response to DNA damage, eukaryotic cells activate a checkpoint signalling cascade, resulting in cell cycle arrest, stabilisation of replication forks and activation of repair. While many players in these pathways have been identified, little is known about the original sensors, or of the DNA structures involved. Because it is present in all checkpoint-inducing lesions, single-stranded DNA (ssDNA) is a good candidate for a common structure recognised by the DNA damage response. The role of ssDNA in checkpoint activation in the yeast Saccharomyces cerevisiae was investigated using three different approaches. Firstly, an attempt was made to produce ssDNA independently of strand breaks by inducing replication-independent plasmid unwinding. Secondly, the effects of depleting the major ssDNA-binding complex, replication protein A (RPA) were analysed. Lastly, an assay to quantify ssDNA generated at a defined DNA double-strand break (DSB) was developed. Despite extensive efforts, the first approach proved unsuccessful, as the method used did not generate unwound plasmid. Using the second approach, it was found that depletion of RPA did not inhibit checkpoint activation during replication stress. Furthermore, replication with limiting amounts of RPA led to rapid cell death and checkpoint activation that was mediated independently of the response to stalled replication forks. Lastly, at a defined DSB it was found that less ssDNA was being generated than had previously been estimated from results based on non-quantitative methods. Additionally, an element of dose dependency was observed in the checkpoint response to DSBs, with stronger and more rapid responses being generated by higher numbers of breaks. Formation of four DSBs resulted in checkpoint activation even in G1 arrested cells. Together, these results raise the possibility of a DNA damage checkpoint pathway largely independent of long tracts of RPA-coated ssDNA and show that checkpoint activation to DSB-damage is possible in G1

Investigating mechanisms and indicators of sensitivity to replication stress-targeting therapies in glioblastoma

Introduction Evidence suggests a subpopulation of treatment resistant glioblastoma (GBM) cancer stem cells (GSCs) is responsible for tumour recurrence, an almost universally deadly characteristic of this cancer of extreme unmet need. Current treatments fail to eradicate GSCs and novel GSC targeting therapies are a clinical priority. Elevated DNA replication stress (RS) in GSCs has been described, leading to constitutive DNA damage response activation and treatment resistance and targeting RS with combined ATR and PARP inhibition (CAiPi) has provided potent GSC cytotoxicity. Nevertheless, there are a relative lack of studies investigating the underlying mechanisms of response to CAiPi in GBM and a lack of robust transcriptional signatures or genomic biomarkers correlated with CAiPi response in GSCs. Aims This thesis aims to investigate RS as a targetable vulnerability of GSCs. It aims to achieve this by studying the mechanisms of sensitivity to inhibition of the RS response to inform transcriptional indicators of sensitivity. Lastly, it aims to investigate the feasibility of this therapeutic strategy in a preclinical model. Methods Paired GSC-enriched and GSC-depleted, differentiated (‘bulk’) populations, derived from resected GBM specimens, were maintained in serum-free, stemenriching conditions or differentiating conditions respectively. WGS and RNAseq were utilised to characterise the genomic and transcriptomic landscape of the cell line panel. Responses to CAiPi were assessed by clonogenic and cell viability assays and validated in a CD133 sorted population by neurosphere assay. Replication dynamics in paired GSC and bulk cells were investigated by a DNA fibre assay. Dysregulated S phase was analysed by quantification of 53BP1 nuclear bodies (53BP1NB), indicative of under-replication of the genome, and quantification of re-replicating cells by flow cytometry. Chromosomal instability was interrogated by quantification of chromatin bridges and micronuclei. Novel mechanistic discoveries prevalent in GSCs with potent CAiPi-sensitivity were used to curate a transcriptional marker of sensitivity for interrogation in GBM cell lines and in published clinical datasets. Lastly the feasibility of CAiPi was investigated in an in vivo preclinical model, assessing tolerability and tumour penetration. Results CAiPi was potently cytotoxic to a population of GSCs but highly heterogenous responses to CAiPi were observed across a panel of seven paired GSCs and bulk cells. Sensitivity was not predicted by elevated RS in GSCs or any previously defined biomarkers of RS or CAiPi sensitivity. Differential sensitivity was exploited for further investigations which identified transcriptional dysregulation of DNA replication, specifically in a CAiPi-responsive GSC line. Subsequent analysis of DNA replication identified PARPi-induced increase in origin firing, associated with PARP trapping. GSCs with this origin firing phenotype also exhibited an increase in both under-replicated DNA and re-replication in response to CAiPi, with an increase in chromosomal aberrations and instability. A curated transcriptional signature, based on mechanistic discoveries in CAiPisensitive GSCs, predicted GSC sensitivity and identified populations of GBM patients with poor survival who may respond to CAiPi treatment. In vivo studies demonstrated murine blood brain barrier (BBB) penetration of a PARPi and an ATRi with minimal toxicity, however optimal dosing and scheduling remains a challenge. Conclusions We propose that CAiPi-sensitivity is marked by loss of replication coordination leading to chromosomal damage as cells move through S phase. Additionally, we propose a model whereby under-replication and re-replication can occur due to spatial and temporal uncoupling during S phase. Targeting RS via CAiPi represents a promising therapeutic strategy for selectively targeting recurrence driving GSCs to improve clinical outcomes in GBM