RNA-DNA hybrid formation at a bacteriophage T4 replication origin

The bacteriophage T4 replication origins ori(uvsY) and ori(34) each contain two distinct components: a T4 middle-mode promoter that is strictly required for replication and a downstream region of about 50 bp that is required for maximal levels of replication. Here, we present evidence that structure of the downstream region is important for replication initiation. Based on sensitivity to a single-stranded DNA-specific nuclease in vitro the downstream region behaves as a DNA unwinding element. The propensity to unwind is probably important for origin activity in vivo, because replication activity is maintained when the native downstream region is replaced with a heterologous DNA unwinding element from pBR322 in either orientation. We analyzed the origin DNA for possible unwinding in vivo by using potassium permanganate, a chemical that reacts with unpaired pyrimidine bases. The non-template strand, but not the template strand, became hypersensitive to permanganate after T4 infection regardless of whether replication could occur. Strand-specific permanganate hypersensitivity was also observed in artificial origins containing the pBR322 DNA unwinding element in either orientation. Hypersensitivity was only detected when the origin contained a promoter that would be active during T4 infection. Furthermore, the origin transcript itself appears to be necessary for hypersensitivity since insertion of a transcriptional terminator abolishes hypersensitivity downstream of the termination site. Our results strongly suggest that the downstream region functions as a DNA unwinding element during replication initiation, leading to the formation of a persistent RNA-DNA hybrid at the origin.