Identification of Residues in SV40 Large Tumor Antigen Important for DNA Replication and Topoisomerase I Binding

Abstract

Eukaryotic DNA replication is a vital molecular process which is not fully understood. Simian Virus 40 provides an ideal model system. Its genome encodes the protein large T‐Antigen (T‐Ag), which orchestrates the initiation of replication. T‐Ag interacts with host cellular protein Topoisomerase I (Topo I). Topo I binds a T‐Ag monomer at two sites, one near the N‐terminal domain (residues 83–160) and one at the C‐terminal end (resides 602–708). Preliminary data suggests that T‐Ag's J‐domain and hinge region residues 101–109, may be involved in Topo I binding. We generated single point mutations in full‐length T‐Antigen and in a N‐terminal construct that contains residues 62–262. In vitro replication assays identified three mutants, N99S, L103Q, and C105F to be replication deficient. Furthermore, DNA replication was not stimulated by the addition of Topo I, as is WT T‐Ag. ELISAs showed mutant T‐Ags N99S, E100A, L103Q, F104V, and E107G have a decrease in Topo I binding as compared to the WT fragment. These data indicate that this region is important for Topo I binding and residues N99 and L103 may be necessary for DNA replication. Other assays will be performed to verify that the mutants’ deficiency in DNA replication is due to a defect in Topo I binding and not to other causes. This work is funded by a PHS Grant from the National Cancer Institute to Dan Simmons and a Howard Hughes Medical Institute Scholarship to Krista Neal.