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Allele-specific analysis of DNA replication origins in mammalian cells.

ABSTRACT: The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity.

SUBMITTER: Bartholdy B

PROVIDER: S-EPMC4479011 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Allele-specific analysis of DNA replication origins in mammalian cells.

Bartholdy Boris B Mukhopadhyay Rituparna R Lajugie Julien J Aladjem Mirit I MI Bouhassira Eric E EE

Nature communications 20150519

The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of ...[more]

PMID: 25987481

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Project description:Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.

Dataset Information

Allele-specific analysis of DNA replication origins in mammalian cells.

Publications

Allele-specific analysis of DNA replication origins in mammalian cells.

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