Abstract
Once per cell cycle replication is crucial for maintaining genome integrity. Geminin interacts with the licensing factor Cdt1 to prevent untimely replication and is controlled by APC/C-dependent cell cycle specific proteolysis during mitosis and in G1. We show here that human geminin, when expressed in human cells in culture under a constitutive promoter, is excluded from the nucleus during part of the G1 phase and at the transition from G0 to G1. The N-terminal 30 amino acids of geminin, which contain its destruction box, are essential for nuclear exclusion. In addition, 30 amino acids within the central domain of geminin are required for both nuclear exclusion and nuclear accumulation. Cdt1 overexpression targets geminin to the nucleus, while reducing Cdt1 levels by RNAi leads to the appearance of endogenous geminin in the cytoplasm. Our data propose a novel means of regulating the balance of Cdt1/geminin in human cells, at the level of the subcellular localization of geminin.
Highlights
The licensing inhibitor geminin must be regulated to ensure once per cell cycle replication
HGem Is Excluded from the Nucleus when Ectopically Expressed During the G1 Phase—Endogenous human geminin localizes to the nucleus, is detected exclusively during S and G2 phases of the cell cycle [21, 22], and is regulated both transcriptionally [23] and by anaphase-promoting complex/cyclosome (APC/C)-mediated proteolysis during G1 [6]
This is consistent with the results of Boos et al [18] who reported that following transfection in human cells, human geminin is less able to localize to the nucleus than Xenopus geminin
Summary
The licensing inhibitor geminin must be regulated to ensure once per cell cycle replication. In Xenopus egg extracts, part of the endogenous geminin escapes degradation in anaphase [14] This surviving population of geminin does not associate with Cdt and does not inhibit licensing [14], and this requires APC/C dependent ubiquitination of geminin [15, 16]. Studies in Xenopus egg extracts suggest that nuclear import of endogenous geminin re-enables it to bind Cdt and inhibit further origin licensing [14], but is important to prevent re-replication during G2 [17]. We suggest that regulation of geminin through changes in its subcellular localization provides a fail-safe mechanism for ensuring a tight balance of Cdt and geminin in the nucleus, thereby controlling timely licensing
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