Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

Abstract

In eukaryotes, initiation of DNA replication requires the activity of the origin recognition complex (ORC). The largest subunit of this complex, Orc1p, has a critical role in this activity. Here we have studied the subnuclear distribution of the overexpressed human Orc1p during the cell cycle. Orc1p is progressively degraded during S-phase according to a spatio-temporal program and it never colocalizes with replication factories. Orc1p is resynthesized in G1. In early G1, the protein is distributed throughout the cell nucleus, but successively it preferentially associates with heterochromatin. This association requires a functional ATP binding site and a protein region partially overlapping the bromo-adjacent homology domain at the N-terminus of Orc1p. The same N-terminal region mediates the in vitro interaction with heterochromatin protein 1 (HP1). Fluorescence resonance energy transfer (FRET) experiments demonstrate the interaction of human Orc1p and HP1 in vivo. Our data suggest a role of HP1 in the recruitment but not in the stable association of Orc1p with heterochromatin. Indeed, the subnuclear distribution of Orc1p is not affected by treatments that trigger the dispersal of HP1.

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

“…9A). The results restricted the NLS to residues 240 -288, a region in which previous studies had identified a NLS at residues 259 -266 (PGRIKRKV) in transient expression assays (40). Therefore, this sequence was changed from RIKRK to GLEGV, and the resulting recombinant FH-Orc1(ϪNLS) protein was 1-6).…”

“…Therefore, Orc1 is translocated to the nucleus where it subsequently associates with other ORC subunits. In early G 1 , Orc1 is distributed throughout the cell nucleus, but as cells progress through late G 1 and S-phase, Orc1 preferentially associates with heterochromatin, a phenomenon associated with amino acids 151-269 (40).…”

Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization

“…Furthermore, Orc2 biochemically interacts indirectly with the heterochromatin protein 1 (HP1) in mammalian, Drosophila, and Xenopus cells (14,15,31,32). Depletion of Orc2 or Orc3 in HeLa cells results in a mitotic arrest with abnormally condensed chromosomes, lack of chromosome alignment at the metaphase plate, and spindle defects (15).…”

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.…”

“…As a result, ORC1 blocks Cyclin E-dependent centriole division, providing for a single round of centrosome duplication. Moreover, it is essential that the activity of the Cyclin E/CDK2 complex is kept inhibited until the early S phase, when Cyclin E is completely degraded, which coincides with ORC1 degradation through the ubiquitin-dependent pathway [47]. It should be noted that the mechanism of ORC involvement in centrosome duplication requires further investigation and the role of the other ORC subunits, which were detected at the centrosomes, still remain obscure.…”