DNA polymerase template switching at specific sites on the φ29 genome causes the in vivo accumulation of subgenomic φ29 DNA molecules

Abstract

SummaryThe accumulation of subgenomic phage 29 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 29 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): 5Ј-TGTTtCAC-GTGg-AACA-3Ј being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the 29 genome. In addition, the almost identical sequence 5Ј-TgGTTTCAC-GTGGAAt-CA-3Ј was found once. These five DSEs are all located in the right half of the 29 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 29 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.

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

“…Another possible mechanism could be that the polymerase double-backs onto its own strand, forming a hairpin. Although we cannot rule this mechanism out, we believe, line with previous findings about replication anomalies (23,24), that a strand drift mechanism is the more plausible explanation.…”

“…We observed that only by using single-stranded DNA binding protein (in this case, T4 gene-32) during RCA with phi29 polymerase, were we able to consistently get single-stranded products. Here we report on the results following this observation and show that a model in which the polymerase shifts from its circular template to its displaced single-stranded DNA, in a similar fashion suggested to sometimes occur in phi29 viral replication (23,24), can explain an occurrence of large amounts of double-stranded DNA following a RCR process.…”

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

“…Our results are consistent with base-pairing-independent copy-choice RNA recombination (1). The ability to switch templates in vitro has been reported for several polymerases (14,15,(27)(28)(29) and thus is a universal activity of template-dependent polymerases. Being able to observe the products of template switch in a simple viral RNA synthesis assay will help elucidate the mechanism for RNA recombination in vitro.…”

Ligation Overcomes Terminal Underrepresentation in Multiple Displacement Amplification of Linear DNA

“…Moreover, they generally disappear in lane 3 (i.e., when digesting the unligated / Hin dIII fragments). In the light of data on the enzymatic properties of the 29 DNA polymerase (12), a reasonable hypothesis is that the unexpected bands are due to the presence of hairpins in the DNA template. These structures are known to cause polymerase detachment from the template, template switching, or specific endonucleolytic activity (13,14).…”

“…NC_001416) and located 3810 bp downstream of the 37459 / Hin dIII site. It is related to the sequence TGTTTCACGTGGAACA, which Murthy et al (12) call a dyad symmetry element (DSE) and show to be specifically responsible for the replication block of the 29 DNA polymerase. This hairpin may thus be responsible for the production of the cited band in lanes 2 and 4 of the Hin dIII digestion (Figure 1A).…”