Effect of a stem-loop structure within the SV40 replication origin upon SV40 T antigen binding to origin region sequences.

Heteroduplexes were formed between SV40 replication origin-containing DNA fragments derived from wild-type genomic DNA and a viral deletion mutant (dl295) lacking 18 base pairs from a 27 bp inverted repeat that is imbedded within the minimal replication origin sequence. Among these structures were molecules bearing a 7 bp stem and a 3 nucleotide loop on the wild-type strand between nucleotides 5230 and 6. By nondenaturing gel electrophoresis, heteroduplex molecules bearing such a structure on the E-strand could be separated from those with the complementary structure on the L-strand, and either heteroduplex could be so separated from either the wild-type or dl295 homoduplex fragment. Each of these forms was singly 5' or 3' end-labeled and then evaluated as a target for specific binding to purified SV40 large T antigen isolated from wild-type virus-infected cells. The results of DNAase footprint protection analyses showed that the existence of both the E- and L-strand stem-loop structures inhibited T binding to site 2 which composes much of the minimal origin sequence. By contrast, T bound readily to this site in both homoduplexes. Furthermore, T protected both E- and L-strand sequences of its strongest binding site (site 1)--which abuts the early side of site 2--when the site 2 stem-loop was on the L-strand, and protected L-strand site 1 sequences normally when the stem-loop was on the E-strand. However, a marked alteration of binding to site 1 E-strand sequences was noted when the stem-loop was on the E-strand. Thus this alteration in replication origin secondary structure resulted in discrete local and vicinal effects on T binding. Furthermore, the results suggest that within site 1--a sequence employed as an early transcriptional regulatory locus--T antigen can bind specifically and tightly to one strand without exhibiting similar behavior on the other strand.