Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

Abstract

SV40 large T antigen (T-ag)is a multifunctional protein that successively binds to 5-GAGGC-3 sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequencespecific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double-and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5-GAGGC-3 sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ϳ10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

Analysis of the Costructure of the Simian Virus 40 T-Antigen Origin Binding Domain with Site I Reveals a Correlation between GAGGC Spacing and Spiral Assembly

“…3A, via K228, N210, and R204). These backbone interactions were, however, also observed in previous studies of the T-ag OBD-GAGGC interaction (24,81). Therefore, these findings are consistent with the proposal that the site I spacer does not function with the pentanucleotides to form an extended recognition Values describing the relationships between the OBDs (i.e., the translation and angular rotation) are indicated.…”

Phosphorylation of Merkel Cell Polyomavirus Large Tumor Antigen at Serine 816 by ATM Kinase Induces Apoptosis in Host Cells

“…Unfortunately, MCV LT S816E behaved identically to the alanine mutant and therefore was not a viable phosphomimetic (data not shown). This phenomenon has occasionally been reported for other phosphoproteins, including SV40 LT and MCV LT, at other phosphosites (34,51). Taken together, these data suggest that phosphorylation of LT at Ser-816 contributes to growth arrest and apoptotic induction mediated by the C-terminal domain.…”

A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication

“…E1 fragments were produced as GST fusion proteins in Escherichia coli BL21(DE3) (Novagen) as previously described (47). GST-E1 fusion proteins were purified and cleaved with thrombin to remove the GST moiety and further processed as described previously (20). All protein concentrations were determined by Bradford analysis.…”