Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

Abstract

The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier–Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1–MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

Distinct and sequential re-replication barriers ensure precise genome duplication

“…We expect that this alteration compromises phosphorylation at most/all CDK-dependent phosphorylation sites. As we had noted in a previous study [28], Cdt1-Cy sometimes accumulated to higher levels than Cdt1-WT, particularly after longer induction times (e.g. Fig 1E, lane 6), and this variant induced the highest amount of both rereplication and Chk1 phosphorylation ( Fig 1B, 1C and 1E).…”

“…Fig 1E, lane 6), and this variant induced the highest amount of both rereplication and Chk1 phosphorylation ( Fig 1B, 1C and 1E). We presume that higher Cdt1-Cy stability contributes to enhanced re-replication activity, but this effect must be independent of phosphorylation at T29 and S31 since Cdt1-Cy is more stable and more active than both Cdt1-9A and a previously-tested Cdt1 mutant "2A" [28].…”

“…This "linker" region connects the two winged-helix domains of Cdt1 that have been characterized for MCM binding (C-terminal "C domain") [26] or for binding to the inhibitor Geminin (middle "M domain") [27]. Both domains are required for metazoan licensing activity [28][29][30][31][32]. We inserted cDNAs encoding either wild-type Cdt1 (Cdt1-WT) or Cdt1-5A into a single chromosomal FRT recombination site under doxycycline-inducible expression control in the U2OS cell line.…”

“…The inhibitory phosphorylation sites are not visible in any currently available Cdt1 atomic structures. Nonetheless, our homology model of the human Cdt1-MCM complex ( [28] and Fig 5A) led us to speculate that phosphorylation-induced changes at this linker could affect MCM binding. We first compared the MCM binding ability of Cdt1-WT to the Cdt1-Cy variant that cannot bind Cyclin A/CDK1.…”

<p>Huaier Suppresses the Hepatocellular Carcinoma Cell Cycle by Regulating Minichromosome Maintenance Proteins</p>

“…A related study reported that knockdown of MCM proteins could induce a delay in S/G2-phase progression by restraining the expression levels of CDK4, CyclinD1, and CyclinE in HCC cells. 28,29 Additionally, MCMs are regulated in the cell cycle through phosphorylation by ATM (ataxia telangiectasia mutated), and phosphorylation of MCM proteins is closely associated with oncogenesis and cancer progression. 30 Importantly, recent studies also demonstrated that both CDK and ATM were regulated by Huaier and finally inhibited cancer cell proliferation.…”