Figure 5.

DNA bridging by BAF and its potential role in blocking autointegration. Linearized double-stranded øX174 DNA (PstI digest of øX174 replicative form I DNA, 30 μM in nucleotides) was preincubated with various concentrations of BAF (0–0.8 μM of monomers) for 10 min on ice. (A) Aggregation of DNA by BAF. One aliquot of each preincubation mixture was centrifuged at 15,000 × g for 10 min. After addition of SDS, the DNA remaining in the supernatants was analyzed by agarose gel electrophoresis and stained with ethidium bromide. (B) Binding of BAF can make a target DNA unavailable for integration. INCs were added to a second set of preincubation mixture aliquots (lanes 2–6) and assayed for their integration activity into the preincubated target DNA. Intermolecular integration reaction products (P₁, ≈14 kbp) were separated from unreacted substrate (S, ≈9 kbp) by agarose gel electrophoresis and visualized by Southern blotting and hybridization. (C) Naked DNA is a preferred target for integration. Linear 2.7-kbp DNA (BstNI digest of øX174 replicative form I DNA, 30 μM in nucleotides) was added to a third set of aliquots of linear 5.4-kbp øX174 DNA that had been preincubated with the various concentrations of BAF. Integration reactions were initiated by addition of INCs (lanes 2–6) and analyzed as described in B except that P₂ (≈12 kbp) represents integration into the naked target DNA. Lanes 1 for B and C are INCs without addition of target DNA. (D) Intermolecular aggregation of DNA by BAF. Bridging of the DNA by BAF results in the formation of a network. (E) Intramolecular bridging by BAF may compact the viral DNA.