To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (-30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase a-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species.Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.With the exception of a single virally encoded protein termed T antigen (T-ag), replication of simian virus 40 (SV40) is dependent on simian or human host proteins (47). The dependence of SV40 replication on host proteins, and the presence of a single, well-defined origin of replication, has made SV40 a useful model system for studies of DNA replication in higher eukaryotes.An in vitro SV40 replication system was developed by Li and Kelly (28), using simian extracts, and extended to human extracts by Stillman and Gluzman (44) and Wobbe et al. (54). This system has enabled the identity and function(s) of many of the host proteins required for SV40 DNA replication to be established (for reviews, see references 8, 22, and 43). It has also permitted an analysis of the mechanisms operating during various stages of the SV40 DNA replication to be studied in detail. We have used this system to study the T-ag-dependent initiation of SV40 DNA synthesis (4, 5).The role played by T-ag during initiation of SV40 DNA replication has been reviewed recently (la, 18). In summary, T-ag recognizes GAGGC binding sites within the SV40 origin, assembles as a double hexamer (30), and melts the early palindrome within the SV40 origin (2). In the presence of topoisomerase I and a single-stranded binding protein, such as the three-subunit complex present in humans (HSSB, also termed RFA or RPA) (8,22,43), the inherent helicase activity of T-ag (42) catalyzes further unwinding of the origin region (11,12,55). T-ag-dependent unwinding of the SV40 origin is a critical initiation event, since it allows additional replication factors to gain entry to the origin region. Less is known about subsequent steps during initiation of SV40 DNA replication.We have conducted pulse-labeling experiments designed to more fully characterize postunwinding initiation events.

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action,showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.

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