DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules.

Abstract

The accumulation of subgenomic phage phi29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis 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 right end of the linear Bacillus subtilis phage phi29 genome. The nature 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 hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules 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 molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGg-AACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the phi29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the phi29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene 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 subgenomic phi29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.