![]() However, whether this happens at origins or at heterochromatin remains to be resolved. We have previously identified ORCA as an ORC-associated protein that can stabilize ORC on chromatin ( 15). Recently, RepID protein was shown to bind to a subset of origins and regulate replication ( 14). Factors including HMGA1 ( 11) or specific histone modifications ( 12, 13) have been implicated in facilitating ORC binding to chromatin. The Drosophila insulator protein Su(Hw) has been shown to facilitate the loading of pre-RC proteins to origins at repressive chromatin regions ( 10). For example, human Myc protein has been shown to interact with ORC and regulate the licensing of lamin B2 origin ( 9). Multiple lines of evidence suggest that in metazoan cells, the selection of an origin and recruitment of pre-RC complex is facilitated by different pre-RC interacting proteins with different DNA binding preferences. In metazoans, however, no consensus sequence at replication origins has been identified and it remains to be determined as to how origins are selected and how ORC is recruited to origins. In Schizosaccharomyces pombe, origins are AT rich but do not appear to have a consensus sequence ( 8). In Saccharomyces cerevisiae, origins are defined by a consensus sequence that is required for ORC binding ( 7). The MCM–Cdc45–GINS (CMG complex) helicase complex is subsequently activated by cell cycle kinases to start replication ( 5, 6). After the loading of MCM2-7, the origin becomes ‘licensed’ and will fire during S phase ( 3). ORC loading results in the sequential recruitment of Cdc6, Cdt1 and the DNA helicase MCM2-7 complex. In eukaryotic cells, this process starts with the loading of a six-subunit complex, Origin Recognition Complex (ORC) at origins during G1 phase ( 3, 4). The initiation of DNA replication requires the step-wise assembly of pre-replication complex (pre-RC) onto origins during G1 ( 3). In eukaryotic cells, DNA replication initiates from multiple distinct sites on each chromosome, which are called replication origins ( 1, 2). Each cell needs to replicate the entire genome once and only once per cell cycle, and this process is regulated both temporally and spatially. We propose that ORCA coordinates with the histone and DNA methylation machinery to establish a repressive chromatin environment at a subset of origins, which primes them for late replication.Įukaryotic cell cycle progression is a highly orchestrated process in which DNA replication, chromatin organization, and transcription need to be precisely coordinated. Furthermore, repressive chromatin marks influence ORCA's binding on chromatin. Similarly, DNA methylation is altered at ORCA-occupied sites in cells lacking ORCA. Regions that associate with both ORCA and H3K9me3, exhibit diminished H3K9 methylation in ORCA-depleted cells, suggesting a role for ORCA in recruiting the H3K9me3 mark at certain genomic loci. ![]() Further, ORCA directly associates with the repressive marks and interacts with the enzymes that catalyze these marks. ![]() The majority of the ORCA-bound sites represent replication origins that also associate with the repressive chromatin marks H3K9me3 and methylated-CpGs, consistent with ORCA-bound origins initiating DNA replication late in S-phase. ORCA association to specific genomic sites decreases as the cells progressed towards S-phase. ORCA binding sites on the G1 chromatin are dynamic and temporally regulated. Here, we evaluated the genome-wide distribution of ORCA using ChIP-seq during specific time points of G1. The ORC-associated protein (ORCA/LRWD1) stabilizes ORC on chromatin. DNA replication requires the recruitment of a pre-replication complex facilitated by Origin Recognition Complex (ORC) onto the chromatin during G1 phase of the cell cycle.
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