Figure 3.

ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb ³²P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. DNA was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb ³²P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded ϕX174 DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.