Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Abstract

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment.

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase CRL4CDT2 Ensures Robust Transition from S Phase to Mitosis

“…At least three substrates (CDT1, p21, and SET8) have known roles in mitosis (21)(22)(23)64), and it is possible that other CRL4 CDT2 substrates also have mitotic roles, making general substrate stabilization important for mitosis. Independent of these mitotic roles, the known substrates of replication-cou-FIGURE 6.…”

“…We show here that CDK1 activity (directly and/or indirectly) inhibits CRL4 CDT2 activity itself by preventing its accumulation on chromatin, an event necessary for CRL4 CDT2 activity. Activation of CDK1 as S phase completes is necessary for the normal reaccumulation of substrates, such as CDT1 and SET8, and we show that, like CDT1, failure to reaccumulate SET8 de novo prior to mitosis leads to mitotic progression defects (21). The temporal control of CRL4 CDT2 activity ensures the accumulation of CRL4 CDT2 substrates during mitosis, thereby preventing chromosome instability.…”

“…1). Importantly, CDT1, p21, and SET8 also function during mitosis, making their reaccumulation critical for normal mitotic progression (21)(22)(23). Replication-coupled destruction is triggered by the ubiquitin E3 ligase, CRL4…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…The loop is required to recruit the Dam1 complex to kinetochores in vivo (Maure et al 2011), but it is not necessarily a direct binding site and the requirement may be due to a structural change that occurs when the loop is deleted. In other organisms, the loop has been implicated in interacting with the Ska1 complex, the Dis1/TOG/Stu2 protein, and the Cdt1 replication factor (Hsu and Toda 2011;Varma et al 2012;Zhang et al 2012), so its precise role is unclear.…”

Genome-scale prediction of moonlighting proteins using diverse protein association information

“…The second example is DNA replication factor Cdt1 (UniProt ID: Q9H211) (Varma et al, 2012). Besides its primary function as DNA replication factor, this MP's secondary function is a role in mitosis where it localizes to kinetochores through binding to the Hec1 component of the Ndc80 complex.…”