EL RECOGNITION SEQUENCES IN THE BOVINE PAPILLOMAVIRUS TYPE-1 ORIGIN OF DNA-REPLICATION - INTERACTION BETWEEN HALF SITES OF THE INVERTED REPEATS

The E1 protein encoded by bovine papillomavirus type 1 (BPV-1) is requ ired for viral DNA replication, and it binds site specifically to an A /T-rich palindromic sequence within the viral origin of replication. T he protein is targeted to this site through cooperative interactions a nd binding with the virus-encoded E2 protein. To explore the nature of the E1 binding site, we inserted a series of homologous DNA linkers a t the center of dyad symmetry within the E1 recognition palindrome. Th e effects of these modifications indicated that the E1 recognition pal indrome can be separated into functional half sites. The series of ins ertions manifest a phasing relationship with respect to the wild-type BPV-1 genome in that greater biological activity was measured when ful l integral turns of the DNA helix separated the palindrome than when t he separations were half-turns. This phasing pattern of activity was o bserved to occur in a variety of biological phenotypes, including tran sformation efficiency, stable plasmid copy number in cell lines establ ished from pooled foci, and transient replication of full-length viral genomes. For replication reporter constructs where E1 and E2 are supp lied in trans by the respective expression vectors, distance between t he half sites seems to play a major role, yet the phasing relationship s are measurable. DNase I protection studies showed that E1 bound very poorly to the construct containing a 5-bp linker, and binding was clo se to the wild-type level for the 10-bp insertion, consistent with a r equirement for a phasing function between half sites with a modulus of 10 bp, Binding to the 15- and 20-bp insertion mutants was weak, but o nly for the 20-bp insertions was protection over both halves of the pa lindrome measurable. As it has been previously reported that the 18-bp palindrome contains sufficient nucleotide sequence information for E1 binding, we speculate that a minimal El recognition motif is presente d in each half site. A comparison between this sequence and that of an upstream region that also binds El (the E2RE1 region) revealed a comm on pentanucleotide motif of APyAAPy. Mutants with substitutions of the ATAAT elements within E2RE1 failed to bind Fl protein. We present mod els for how repeats of the pentanucleotide sequence may coordinate E1 binding at the dyad symmetry axis of the origin and compare the DNA se quence organization of BPV-1 with those of the simian virus 40 and pol yomaviruses at their origins of DNA replication.