Figure 3

The Mpn SSB protein binds oligonucleotide substrates in a DNA sequence-independent fashion. (A) Binding of Mpn H₁₀-SSB to three different oligonucleotide substrates. Reactions were performed in volumes of 20 μl and contained either 0 μM (lanes 2, 6 and 10), 1.3 μM (lanes 3, 7 and 11), 3.1 μM (lanes 4, 8 and 12) or 9.4 μM (lanes 5, 9 and 13) of Mpn H₁₀-SSB, and 5 μM of either of three different single-stranded oligonucleotides (Oligo 1, 2 or 3; Table 1). After incubation for 15 min at 37°C, the samples were electrophoresed on a 1.0% agarose gel in 0.5 × TBE buffer. A black/white inverted image of a typical ethidium bromide-stained gel is shown. (B) Binding of Mpn H₁₀-SSB (at 0, 1.3, 3.1 or 9.4 μM in lanes 1–4, 5–8, 9–12 and 13–16, respectively) to 5'-³²P-labeled homooligomeric DNA substrates (at 1 μM; Table 1). The samples were separated on 5% polyacrylamide gels in 0.5 × TBE buffer. An autoradiograph is shown. (C) Binding of Mpn H₁₀-SSB (at 0, 1.3, 3.1 or 9.4 μM) to a series of 15- to 50-mer 5'-³²P-labeled oligonucleotides (at 1 μM), each containing the same 15-nucleotide core sequence (Table 1). The samples were processed as described above in (B). (D) A DNA-binding competition experiment in which Mpn H₁₀-SSB (at 3.1 μM) was incubated with a constant amount (1 μM) of either the 5'-³²P-labeled '20 nt' oligonucleotide (lanes 1–7) or '50-nt' oligonucleotide (lanes 8–13) (Table 1), and increasing amounts of the other, unlabeled ('cold') oligonucleotide. A molar excess of 5 to 100 times unlabeled oligonucleotide over labeled oligonucleotide was tested, as indicated above the lanes. From the samples loaded in lanes 1 and 8, Mpn H₁₀-SSB was omitted; the gel was processed similarly as in (B).