Figure 4

Mpn SSB binding to long DNA substrates and Mg²⁺-dependence of DNA-binding. (A) Binding of E. coli SSB (Eco SSB, lanes 1–8) and Mpn SSB (lanes 9–16) to either 2 nM of ss φX174 DNA (ssDNA, lanes 1–4 and lanes 9–12) or 1 nM of ds φX174 DNA (dsDNA, lanes 5–8 and lanes 13–16). Reactions were performed in volumes of 20 μl and contained various concentrations of protein, as indicated above the lanes. The samples were separated on a 0.6% agarose gel in 0.5 × TBE buffer. A black/white inverted image of a typical ethidium bromide-stained gel is shown. (B) Binding of GST-SSB to circular, single-stranded M13mp18 DNA (ssDNA). Binding reactions were carried out with various concentrations of either GST-SSB (lanes 8–11) or GST (lanes 3–6), as indicated above the lanes, and 2 nM of DNA. The samples were separated on 0.6% agarose gels in 0.5 × TBE buffer and processed as described above. (C) DNA-binding by Mpn SSB is Mg²⁺-independent. Reactions with Mpn H₁₀-SSB were executed similarly as described in (B), except for the omission of Mg(OAc)₂ in the reaction mixtures of the samples loaded in lanes 2–5. Complexes of Mpn SSB bound to ssDNA (SSB-ssDNA), and the position of unbound DNA (ssDNA and dsDNA), are indicated alongside the gel. The marker DNA loaded in lane 1 of both (B) and (C) is the SmartLadder (Eurogentec).