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Selectivity of ORC binding sites in the human genome and the relation to replication timing, fragile sites, and recurrent deletions in cancers

ABSTRACT: The origin recognition complex (ORC) binds specific sites in mammalian genomes, from which DNA replication is initiated. Here, we address ORC binding selectivity in vivo by mapping ~52,000 ORC2 binding sites throughout the human genome. The ORC binding profile is broader than that of sequence-specific transcription factors, suggesting that it does not bind to specific DNA sequences. Instead, ORC binds to open (DNase I hypersensitive) regions in which histone H3 tails are hyperacetylated and also di-methylated at lysine 4. Our results suggest that ORC binding selectivity in vivo is determined by a novel mechanism involving non-specific interaction with accessible DNA and recognition of modified histones. ORC sites are far more prevalent in early replicating regions, suggesting that replication timing is primarily due to ORC density and stochastic firing of origins. Large genomic regions lacking ORC2 binding sites are strongly associated with common fragile sites and recurrent deletions in cancers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE70165 | GEO | 2016/06/01

SECONDARY ACCESSION(S): PRJNA287782

REPOSITORIES: GEO

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Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.

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Selectivity of ORC binding sites in the human genome and the relation to replication timing, fragile sites, and recurrent deletions in cancers

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