Figure 3.

Specific open ‘4-way’ junctions are good substrates for second-strand DNA cleavage. Various R2Bm/28S derived junctions related to the open 4-way junction drawn in step 3 of the New Integration Model (Figure 1) were tested for DNA cleavage. Symbols, conventions, reactions, gels, analysis and graphs are as in Figure 2. The North arms of the constructs contain 47 bp of 28S downstream DNA, which is the same amount of downstream 28S DNA normally used in our linear target DNA (10,20). In construct xiii the 47 bp North arm was replaced with a 35 bp arm of mostly non-specific DNA. The 5 bp nearest the cleavage site, however, remained 28S DNA. The West arm of constructs v–xii were identical to constructs iii and iv (Figure 2), being 25 bp in length and containing mostly nonspecific DNA. The West arms of constructs xiii–xvi were 73 bp of upstream DNA and corresponds to the amount of upstream DNA normally used in our linear target DNA (6,29). East and South arms of all constructs are 25 bp. See Supplementary Figure S3 for mapping of DNA cleavages. See Supplementary Figure S4 for endonuclease mutant protein controls and for a graph of fraction bound as a function of protein concentration. See Supplementary Figure S5 for denaturing gels of specific EMSA bands.