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Fu H.,U.S. National Institutes of Health | Martin M.M.,U.S. National Institutes of Health | Regairaz M.,U.S. National Institutes of Health | Huang L.,U.S. National Institutes of Health | And 7 more authors.
Nature Communications | Year: 2015

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. Source


Fu H.,U.S. National Cancer Institute | Maunakea A.K.,U.S. National Institutes of Health | Martin M.M.,U.S. National Cancer Institute | Huang L.,U.S. National Cancer Institute | And 6 more authors.
PLoS Genetics | Year: 2013

Mammalian DNA replication starts at distinct chromosomal sites in a tissue-specific pattern coordinated with transcription, but previous studies have not yet identified a chromatin modification that correlates with the initiation of DNA replication at particular genomic locations. Here we report that a distinct fraction of replication initiation sites in the human genome are associated with a high frequency of dimethylation of histone H3 lysine K79 (H3K79Me2). H3K79Me2-containing chromatin exhibited the highest genome-wide enrichment for replication initiation events observed for any chromatin modification examined thus far (23.39% of H3K79Me2 peaks were detected in regions adjacent to replication initiation events). The association of H3K79Me2 with replication initiation sites was independent and not synergistic with other chromatin modifications. H3K79 dimethylation exhibited wider distribution on chromatin during S-phase, but only regions with H3K79 methylation in G1 and G2 were enriched in replication initiation events. H3K79 was dimethylated in a region containing a functional replicator (a DNA sequence capable of initiating DNA replication), but the methylation was not evident in a mutant replicator that could not initiate replication. Depletion of DOT1L, the sole enzyme responsible for H3K79 methylation, triggered limited genomic over-replication although most cells could continue to proliferate and replicate DNA in the absence of methylated H3K79. Thus, prevention of H3K79 methylation might affect regulatory processes that modulate the order and timing of DNA replication. These data are consistent with the hypothesis that dimethylated H3K79 associates with some replication origins and marks replicated chromatin during S-phase to prevent re-replication and preserve genomic stability. Source


Martin M.M.,NCI Inc | Ryan M.,InSilico Solutions | Kim R.,InSilico Solutions | Zakas A.L.,NCI Inc | And 12 more authors.
Genome Research | Year: 2011

This report investigates the mechanisms by which mammalian cells coordinate DNA replication with transcription and chromatin assembly. In yeast, DNA replication initiates within nucleosome-free regions, but studies in mammalian cells have not revealed a similar relationship. Here, we have used genome-wide massively parallel sequencing to map replication initiation events, thereby creating a database of all replication initiation sites within nonrepetitive DNA in two human cell lines. Mining this database revealed that genomic regions transcribed at moderate levels were generally associated with high replication initiation frequency. In genomic regions with high rates of transcription, very few replication initiation events were detected. High-resolution mapping of replication initiation sites showed that replication initiation events were absent from transcription start sites but were highly enriched in adjacent, downstream sequences. Methylation of CpG sequences strongly affected the location of replication initiation events, whereas histone modifications had minimal effects. These observations suggest that high levels of transcription interfere with formation of pre-replication protein complexes. Data presented here identify replication initiation sites throughout the genome, providing a foundation for further analyses of DNA-replication dynamics and cell-cycle progression. Source

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