Human Cdt1 Lacking the Evolutionarily Conserved Region That Interacts with MCM2–7 Is Capable of Inducing Re-replication

Jamie K Teer,Anindya Dutta

doi:10.1074/jbc.m708767200

Abstract

Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2-7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2-7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin.

Highlights

DNA replication is a complex process that a cell must perform accurately to ensure the genome is copied correctly with minimal damage
cyclin-dependent kinase (CDK) activity is low in G1, when the pre-replication complex is loaded
MCM2–7 bridges the two CDK activity states that function to license replication: its chromatin loading must occur in low CDK activity, and its activation and origin firing must occur in high CDK activity

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—293T cells, human embryonic kidney cells transformed with adenovirus oncogenes E1a and E1b and with simian virus 40 oncogene T antigen, were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% iron-supplemented donor calf serum and 1% penicillin/streptomycin. Western Blotting, Immunoprecipitation, and siRNA—Western blotting was performed as described In this case, cells were lysed in 50 mM Tris, pH 7.4, 0.2% Nonidet P-40, 150 or 300 mM NaCl, 1 mM EDTA, 10 mM NaF, 0.2 mM Na3VO3, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, and a 1:100 protease inhibitor mixture (Sigma). Chromatin Fractionation—Chromatin fractionation protocols used to observe MCM7 and Cdt chromatin loading were described previously Pellets were incubated in 100 ␮l of lysis buffer containing 1 mM CaCl2 and 120 units of micrococcal nuclease (Worthington) for 10 min at 37 °C and centrifuged. 0.1% Triton X-100 was added, and the cells were incubated for 5 min on ice. Nuclei were collected in pellet 1 (P1) by low-speed centrifugation (4 min, 1,300 ϫ g, 4 °C). Nuclei were collected by low-speed centrifugation and lysed as above [27]

RESULTS

Proteins not solubilized by MNase treatment remain in the insoluble

DISCUSSION