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

Abstract

The origin recognition complex (ORC) binds sites from which DNA replication is initiated. 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 those of sequence-specific transcription factors, suggesting that ORC is not bound or recruited to specific DNA sequences. Instead, ORC binds nonspecifically to open (DNase I-hypersensitive) regions containing active chromatin marks such as H3 acetylation and H3K4 methylation. ORC sites in early and late replicating regions have similar properties, but there are far more ORC sites in early replicating regions. This suggests that replication timing is due primarily to ORC density and stochastic firing of origins. Computational simulation of stochastic firing from identified ORC sites is in accord with replication timing data. Large genomic regions with a paucity of ORC sites are strongly associated with common fragile sites and recurrent deletions in cancers. We suggest that replication origins, replication timing, and replication-dependent chromosome breaks are determined primarily by the genomic distribution of activator proteins at enhancers and promoters. These activators recruit nucleosome-modifying complexes to create the appropriate chromatin structure that allows ORC binding and subsequent origin firing.DNA replication | replication origins | chromatin | replication timing | ORC R eplication origins are established by the assembly of the prereplication complex at discrete sites of the genome. The first step of this process involves binding of the highly conserved six-subunit origin recognition complex (ORC), which serves as a loading platform for the subsequent assembly of helicases, DNA polymerases, and cofactors required for DNA synthesis (1, 2). In the yeast Saccharomyces cerevisiae, ORC binds DNA in an ATP-dependent manner and recognizes a specific DNA sequence (3). In Drosophila, ORC localizes to regions of open chromatin with contributions from activating histone modifications, DNA sequence, DNA binding proteins, and nucleosome remodelers (4-6). In mammals, the mechanism(s) through which ORC is localized and establishes a functional origin remains unclear.A great deal of effort and a variety of experimental approaches have been devoted to describing the nature and position of replication origins in mammalian genomes. DNA combing technology, replication timing analysis, short nascent strand (SNS) enrichment, and bubble trapping approaches suggest that DNA replication initiation sites are enriched in CpG-rich regions, open chromatin domains, and transcriptional regulatory elements (7-17). However, these methods lack the necessary resolution to investigate important relationships of ORC binding with other features of the genome. In addition, the divergence in protocols and bioinformatic pipelines between laboratories has led to some controversial and nonreproducible observations. Finally, these studies assume that the identified replication initiation sites are compa...

Nonreplicative functions of the origin recognition complex

“…In particular, ORC-binding sites coincide with DNAse I hypersensitive sites and histone modifications of active chromatin [87–92]. The majority of the ORC binding sites coincide with transcription regulatory elements, such as promoters and transcriptional enhancers [85]. The preference of ORC to bind to open chromatin also explains a high correlation between the ORC binding sites and the binding sites of various transcription factors, which has been described previously [86,93,94].…”

“…According to the latest data, ORC has a large number of binding sites throughout the genome: about 5,000 in Drosophila [77] and about 52,000 in mammals [85]. The analysis of the origin activity revealed an existence of more than 80,000 of origins that were active in various human and mouse ESC lines.…”

“…In a recent study [85], mathematical simulations of replication (during the S phase of the cell cycle) have shown that the firing of replication origins is, most likely, a stochastic process. According to this model, frequent distribution of ORC binding sites in the euchromatin results in its frequent firing by chance and, therefore, in the earlier replication of the whole region, compared to heterochromatic regions [85].…”

“…According to this model, frequent distribution of ORC binding sites in the euchromatin results in its frequent firing by chance and, therefore, in the earlier replication of the whole region, compared to heterochromatic regions [85]. This model explains not only the existence of the early- and late-replicating regions, due to the irregular distribution of ORC binding sites throughout the genome, but also their correlation with eu- and heterochromatin.…”

Comment l’approche génomique aide à comprendre le processus d’initiation de la réplication

“…Ainsi, l'initiation de la réplication se produit dans des régions (ou zones) particulières et de manière bidirectionnelle. Les origines de réplication sont souvent associées à des éléments régulateurs de la transcription, comme les promoteurs et les séquences cis-régulatrices, suggérant que les mécanismes impliqués dans la régulation de la transcription pourraient également s'appliquer à la régulation de l'initiation de la réplication [4][5][6][7][8][9][10]. Une analyse plus approfondie des données démontre, toutefois, que les mécanismes de recrutement du complexe ORC et d'initiation de la réplication diffèrent d'une espèce à l'autre [3].…”