Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance

Abstract

The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1α (HP1α) and HP1β in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle

Nonreplicative functions of the origin recognition complex

“…ORC and HP1 co-localization on heterochromatin was also demonstrated in the mammalian cells [43–47]. Nevertheless, it is still unclear whether ORC interact with HP1 on the same regions of heterochomatin, because depletion of the individual ORC subunits in human cells has different outcomes on HP1 localization on heterochromatin.…”

“…It has been shown that ORC2 localizes to the centromeres during the cell cycle G2/M phase [43,54,55] (Figure 1a). Post-translational modification of ORC2 with the SUMO2 attachment is important for the maintenance of centromeric histone H3 methylation status, and the prevention of heterochromatin re-replication via the recruitment of demethylase KDM5A [8] (Figure 1b).…”

“…Upon the completion of cell division each of the daughter cells receives only one centrosome which is further duplicated and reorganized during the interphase to be ready for the next round of division [97]. Immunostaining of mammalian cells revealed the presence of the ORC1–4 proteins at centrosomes [98], and demonstrated that through the cell division, ORC2 localizes at the centromeric regions of chromosomes [43,54,55,98]. In mammalian cells, ORC2 depletion leads not only to defects in the DNA replication, but also to abnormalities during cell division, including noticeable defects in chromosome segregation and centrosome division, however, the precise role of ORC in these processes so far remains unclear [43].…”

“…Immunostaining of mammalian cells revealed the presence of the ORC1–4 proteins at centrosomes [98], and demonstrated that through the cell division, ORC2 localizes at the centromeric regions of chromosomes [43,54,55,98]. In mammalian cells, ORC2 depletion leads not only to defects in the DNA replication, but also to abnormalities during cell division, including noticeable defects in chromosome segregation and centrosome division, however, the precise role of ORC in these processes so far remains unclear [43]. The subsequent studies have found that ORC1 contains the centrosomal localization sequence and is directly involved in the regulation of centrosome division, its role being tightly associated with the functions of cyclins [18,19].…”

Mitotic Functions for SNAP45, a Subunit of the Small Nuclear RNA-activating Protein Complex SNAPc

“…For example, subunits of Orc such as Orc2 and Orc6 localize to centrosomes, centromeres, and heterochromatin in the first case, and kinetochores and mid-bodies in the second case. Down-regulation of these proteins by siRNA causes not only defects in S phase but also multiple centrosomes, abnormally condensed chromosomes, failed chromosome congression, and, in the second case, multinucleated cells (30). As another example, BRCA1 localizes to centrosomes (31,32), and its down-regulation causes amplification and fragmentation of centrosomes in cell lines derived from mammary tissue (33,34) and defective chromosome condensation and segregation in other cell lines (35).…”