Abrogation of a mitotic checkpoint by E 2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p 53 tumor suppressor protein the fission yeast , Schizosaccharomyces pombe , a single

the fission yeast, Schizosaccharomyces pombe, a single Mark G.Frattini1,2, Stephen D.Hurst1, CDK (p34cdc2) regulates cell cycle progression in combinHock B.Lim1, ation with different phase-specific cyclin proteins (Enoch Sathyamangalam Swaminathan1 and Nurse, 1991; Hayles et al., 1994; King et al., 1994; and Laimonis A.Laimins1,3,4 Nurse, 1994). However, in mammalian cells, a series of Departments of 1Microbiology-Immunology and 3Biochemistry, cyclin proteins are synthesized sequentially during the cell Molecular Biology and Cell Biology, Northwestern University, cycle and form active complexes with multiple CDKs 303 E. Chicago Ave, Chicago, IL 60611, USA (Heichman and Roberts, 1994; Grana and Reddy, 1995; 2Present address: The University of Chicago, Pritzker School of Morgan, 1995; Su et al., 1995). The D-type cyclins Medicine, Chicago, IL 60637, USA associate with several CDK proteins in mid-G1, while 4Corresponding author cyclins E and A associate with cdk2 in late G1 and S phase, respectively. The transition from S phase into Human papillomavirus (HPV) E2 and E1 proteins are mitosis and the induction of spindle formation is controlled required for the replication of viral genomes in vivo. by complexes of cyclins A and B with the cdc2 gene We have examined the effects of increasing the level of product (Enoch and Nurse, 1991; King et al., 1994; Nurse, E2 on viral and cellular replication using recombinant 1994). Following completion of each phase of the cell adenoviruses. Infection of cells which maintain HPV cycle, the active complexes are specifically degraded or 31 DNA episomally with E2 recombinant adenoviruses rendered inactive in order to prevent any interference with resulted in a 5-fold increase in genome copy number progression. as well as an S phase arrest allowing for the continued Cell cycle progression is also regulated through negative replication of cellular DNA. Similar effects on cell cycle feedback on the cyclin–CDK activity by inhibitors of the progression were seen following infection of normal CDKs which include p21, p27, p16, p18, p19, p20 human foreskin keratinocytes, the natural host cell. and p57 (Grana and Reddy, 1995; Morgan, 1995). The The DNA content of these cells increased beyond 4N transcriptional activator, p53, senses damage to DNA and, indicating that multiple rounds of replication had in response, activates the expression of the p21 gene occurred without an intervening mitotic event. In product (El-Deiry et al., 1993). This increase in p21 addition, increased cyclin A and E associated kinase protein level inhibits cyclin E–cdk2 and cyclin A–cdk2 activity was observed, while no change was detected activities, resulting in cell cycle arrest. Another important in cyclin B associated kinase activity or in the activation checkpoint exists at the end of S phase, insuring that state of cdc2 kinase. Interestingly, the levels of the p53 DNA replication has been completed faithfully prior to tumor suppresser protein were dramatically reduced progression into mitosis (Enoch and Nurse, 1991; through a post-transcriptional mechanism following Heichman and Roberts, 1994; Nurse, 1994; Su et al., 1995). infection. These data suggest a role for E2 in regulating The process of DNA replication is highly coordinated and viral and cellular replication by abrogation of a mitotic dictates that each replication origin is activated once per checkpoint, which is, at least in part, controlled by p53. cell cycle even though initiation occurs at different times