Figure 4

Preferential binding of dsDNA by SPO-11. Negatively supercoiled dsDNA (sc dsDNA) (a) or linear dsDNA (b) was used as substrate for SPO-11. (c) Binding of the 80 bp dsDNA, its constituent top and bottom ssDNA strands, and 80-mer poly-dT by SPO-11 was tested. The ssDNA binding protein RPA was included in the analysis. (d) The 80 bp dsDNA and top ssDNA strand were co-incubated with the indicated concentration of SPO-11. (e) Complex of SPO-11 and Cy5-labeled dsDNA was challenged with a 1, 5, or 10-fold molar amount of the indicated unlabeled DNA. Note that the agarose gels in panels a and b were stained with ethidium bromide; gels in panels c and d were stained with SYBR Gold to allow better detection of the ssDNA; DNA in the gel in panel e was detected by Cy5 fluorescence. In panels a, b, and d, PK denotes proteinase K.