DNA-POLYMERASE TEMPLATE SWITCHING AT SPECIFIC SITES ON THE PHI-29 GENOME CAUSES THE IN-VIVO ACCUMULATION OF SUBGENOMIC PHI-29 DNA-MOLECULES

The accumulation of subgenomic phage phi 29 DNA molecules with specifi c sizes was observed after prolonged infection times with delayed lysi s phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6,5, 2,3, 2 and 1 kb, Most of the molecules were shown to originate from the ri ght end of the linear Bacillus subtilis phage phi 29 genome, The natur e of the 4, 2,3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming ha irpin structures, The other molecules consisted of palindromic linear double-stranded DNA molecules, Most probably, the subgenomic DNA molec ules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo. Determination of the centres of symmetry of the 4 and 1 kb molec ules revealed that both contained the almost 16 bp perfect dyad symmet ry element (DSE): 5'-TGTTtCAC-GTGg-AACA-3' being a likely candidate fo r a protein binding site. Database analysis showed that this sequence occurs four times in the phi 29 genome. In addition, the almost identi cal sequence 5'-TgGITTCAC-GTGGAAt-CA-3' was found once. These five DSE s are all located in the right half of the phi 29 genome, and the same sequences are also present in the linear DNA of related B, subtilis p hages, Most interestingly, this sequence is also found in the spoOJ ge ne of the B. subtilis chromosome, Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE, As the same su bgenomic phi 29 DNA molecules accumulate after infection of B. subtili s spoOJ deletion strains, it is likely that, in addition to and/or ind ependently of SpoOJ, other protein(s) bind to DSE.