PROTEIN DOMAINS CONNECT CELL-CYCLE STIMULATION DIRECTLY TO INITIATIONOF DNA-REPLICATION

Polyoma large T antigen (LT) is the only viral gene product required f or viral DNA replication. LT can be divided into two domains, one N-te rminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary ce lls 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 repli cation in vivo in growing mouse cells at a level approaching that of f ull-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 ce lls showed that NT was sufficient for inducing S phase, suggesting a m echanism for complementation. By contrast, CT was unable to induce S p hase when tested in the same assay. NT also promotes phosphorylation o f sites in CT that are likely to be important for replication. Other D NA tumor virus gene products such as adenovirus E1A 12S and human papi llomavirus 16 E7 could also complement CT for replication. Although NT , E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p 107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation.