Protein Domains Connect Cell Cycle Stimulation Directly to Initiation of DNA Replication

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 25; pp. 12125 - 12129
• Main Authors: Gjorup, Ole V., Rose, Paul E., Holman, Patricia S., Bockus, Beverly J., Schaffhausen, Brian S.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 06.12.1994; National Acad Sciences; National Academy of Sciences
• Subjects: 3T3 Cells; adenovirus; Adenovirus E1A Proteins - metabolism; Animals; Antigens, Polyomavirus Transforming - metabolism; Binding sites; Biochemistry; Cell Cycle; Cell growth; Cellular biology; Deoxyribonucleic acid; DNA; DNA Replication; Genes, Viral; Harvey murine sarcoma virus - genetics; Harvey murine sarcoma virus - metabolism; Interphase; Mice; Oncogene Proteins, Viral - metabolism; Papillomaviridae - genetics; Papillomaviridae - metabolism; Papillomavirus E7 Proteins; Peptide Fragments - metabolism; Phosphorylation; Plasmids; Proteins; Recombinant Proteins - metabolism; Retinoblastoma Protein - metabolism; Transfection; Virus Replication
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.25.12125

Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation.