Best Practices for Mapping Replication Origins in Eukaryotic Chromosomes

Besnard, Emilie; Desprat, Romain; Ryan, Michaël; Kahli, Malik; Aladjem, Mirit I.; Lemaı̂tre, Jean-Marc

doi:10.1002/0471143030.cb2218s64

Cited by 5 publications

(5 citation statements)

References 68 publications

(167 reference statements)

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“…We also mapped replication initiation sites by short nascent strand sequencing [41,42] and found the presenescent cells had fewer origin peak (Figure 1 increased number of regions without detectable origins ( Figure 1(g)) in pre-senescent cells, compared to proliferative cells at early passages. The detection of an increased origin density by DNA fibers, and fewer detectable initiation sites, by mapping, are both consistent with the activation of many dormant origins, within different cells using different cohorts of origins [34,39,40].…”

Section: Induction Of Replication Stress Upon Entry Into Senescencementioning

confidence: 99%

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Cellular senescence induces replication stress with almost no affect on DNA replication timing

et al. 2018

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Organismal aging entails a gradual decline of normal physiological functions and a major contributor to this decline is withdrawal of the cell cycle, known as senescence. Senescence can result from telomere diminution leading to a finite number of population doublings, known as replicative senescence (RS), or from oncogene overexpression, as a protective mechanism against cancer. Senescence is associated with large-scale chromatin re-organization and changes in gene expression. Replication stress is a complex phenomenon, defined as the slowing or stalling of replication fork progression and/or DNA synthesis, which has serious implications for genome stability, and consequently in human diseases. Aberrant replication fork structures activate the replication stress response leading to the activation of dormant origins, which is thought to be a safeguard mechanism to complete DNA replication on time. However, the relationship between replicative stress and the changes in the spatiotemporal program of DNA replication in senescence progression remains unclear. Here, we studied the DNA replication program during senescence progression in proliferative and pre-senescent cells from donors of various ages by single DNA fiber combing of replicated DNA, origin mapping by sequencing short nascent strands and genome-wide profiling of replication timing (TRT). We demonstrate that, progression into RS leads to reduced replication fork rates and activation of dormant origins, which are the hallmarks of replication stress. However, with the exception of a delay in RT of the CREB5 gene in all pre-senescent cells, RT was globally unaffected by replication stress during entry into either oncogene-induced or RS. Consequently, we conclude that RT alterations associated with physiological and accelerated aging, do not result from senescence progression. Our results clarify the interplay between senescence, aging and replication programs and demonstrate that RT is largely resistant to replication stress.

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Section: Induction Of Replication Stress Upon Entry Into Senescencementioning

confidence: 99%

“…We isolated short nascent strands at replication origins of asynchronous human cells as previously described [41,42]. For each cell type, we purified the short nascent strands of two biological replicates.…”

Section: Isolation Of Short Nascent Strands At Replication Originsmentioning

confidence: 99%

Cellular senescence induces replication stress with almost no affect on DNA replication timing

et al. 2018

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“…Partially unwound DNA are likely to form only in the vicinity of replication origins, and such structures can be mapped by virtue of being branched. For the relatively low throughput of two-dimensional gel agarose electrophoresis, just a small set of activity origins in the smallest chromosomes in S. cerevisiae were located by this method ( Reynolds et al, 1989 ; Newlon et al, 1993 ; Friedman et al, 1997 ; Besnard et al, 2014 ).…”

Section: Experimental Methods To Identify Yeast Replication Originsmentioning

confidence: 99%

“…Some methods can even define replication origin sequences throughout the genome with single-nucleotide resolution. On the other hand, next-generation sequencing technologies exhibit coverage biases, which should be avoided to ensure the accuracy of whole-genome origin maps ( Besnard et al, 2014 ).…”

Section: Experimental Methods To Identify Yeast Replication Originsmentioning

confidence: 99%

Recent advances in the genome-wide study of DNA replication origins in yeast

et al. 2015

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DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve yeast replication origins prediction.

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“…Understanding of how major DNA polymerases, POLE and POLD1, divide the labor during DNA replication has largely stemmed from patterns of mutation or ribonucleotide incorporation in cell systems with deficiencies in different proofreading mechanisms (Nick McElhinny et al 2008;Larrea et al 2010a;Lujan et al 2012;Johnson et al 2015a;Reijns et al 2015;Clausen et al 2015;Andrianova et al 2017). By contrast, a qualitative model of replication has been based on reconstruction of replication in vitro (Yeeles et al 2015;Georgescu et al 2014;Yeeles et al 2017;Kurat et al 2017;Devbhandari et al 2017), and efficiency of replication origins in mammalian cells has been estimated mainly from sequencing of 500-2500 nucleotide long stretches of nascent DNA (Cayrou et al 2011;Besnard et al 2012Besnard et al , 2014Cayrou et al 2015). Analyses of mutagenesis in systems with deficiencies of different components of proofreading machinery have not yet been applied to study firing of replication origins.…”

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confidence: 99%

POLD replicates both strands of small kilobase-long replication bubbles initiated at a majority of human replication origins

et al. 2017

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Error-prone mutants of polymerase epsilon (POLE*) or polymerase delta (POLD1*) induce a mutator phenotype in human cancers. Here we show that the rate of mutations introduced by POLD1* is elevated by 50%, while the rate of POLE*-induced mutations is decreased twofold, within one kilobase from replication origins. These results support a model in which POLD1 replicates both the leading and the lagging strands within a kilobase from an origin. The magnitude of the mutational bias suggests that the probability of an individual origin to initiate replication exceeds 50%, which is much higher than previous estimates. Using additional data from nascent DNA sequencing and Okazaki fragments sequencing (OK-seq) experiments, we showed that a majority of origins are firing at each replication round, but the initiated replication fork does not propagate further than 1Kb in both directions. Analyses based on mutational data and on OK-seq data concordantly suggest that only approximately a quarter of fired origins result in a processive replication fork. Taken together, our results provide a new model of replication initiation. KeywordsPOLE; POLD; replication; origins; MSI; cancer Accumulation of sequencing data is improving the understanding of processes involved in mutagenesis. Recently, analysis of preferential fork direction helped uncover a major mode of APOBEC-induced mutagenesis in cancer (Seplyarskiy et al. 2016a;Morganella et al. 2016;Haradhvala et al. 2016) and revealed the dominant role of mismatches introduced by POLD1 in mutagenesis of cancers with mismatch repair

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Best Practices for Mapping Replication Origins in Eukaryotic Chromosomes

Cited by 5 publications

References 68 publications

Cellular senescence induces replication stress with almost no affect on DNA replication timing

Cellular senescence induces replication stress with almost no affect on DNA replication timing

Recent advances in the genome-wide study of DNA replication origins in yeast

POLD replicates both strands of small kilobase-long replication bubbles initiated at a majority of human replication origins

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