Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication

Abstract

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E–CDK2 and Cyclin A–CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A–CDK2 kinase activity but has no effect on Cyclin E–CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E–CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E–CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…Cyclin E/CDK2 and Cyclin A/CDK2 were expressed in recombinant baculovirus infected Hi5 insect cells and purified as in Hossain and Stillman [48]. The phosphorylation of 1 µg of wild type and T592 mutant his-tagged SAMHD1 was carried out in the presence of 10 nM of purified CyclinA/CDK2 or CyclinE/CDK2 in a 15 µl reaction containing 0.1 mg/ml BSA, 0.2 mM cold ATP and 1 µCi [γ-P 32 ]-labeled ATP in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 1 mM DTT) for 30 min at 37°C.…”

Nonreplicative functions of the origin recognition complex

“…In addition to its participation in several processes within the cell nucleus, ORC has a number of cytoplasmic functions, one of which is an involvement of certain subunits of the complex in the regulation of centrosome duplication [18,19]. …”

“…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]. The precise mechanism of its action, has not yet been described, although it has been established that ORC1 inhibits the kinase activity of the Cyclin A/CDK2 and Cyclin E/CDK2 complexes through its interaction with cyclins [18,99].…”

“…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]. The precise mechanism of its action, has not yet been described, although it has been established that ORC1 inhibits the kinase activity of the Cyclin A/CDK2 and Cyclin E/CDK2 complexes through its interaction with cyclins [18,99]. As a result, ORC1 blocks Cyclin E-dependent centriole division, providing for a single round of centrosome duplication.…”

“…In the last few decades, involvement of ORC in numerous highly diverse cellular functions was discovered, e. g. the formation of heterochromatin, chromosome condensation and segregation, centrosome duplication, cytokinesis and mRNA nuclear export [14–17]. The sub-complexes or the individual subunits of ORC are localized not only in nuclei and on the mitotic chromosomes, but in the cytoplasm as well [18–21]. Interestingly, ORC subunits have also been detected in terminally differentiated mammalian cells that do not undergo subsequent cell divisions, for example, cardiomyocytes and neurons [22].…”