Fig 2.

Summary of breakpoint locations for substrate pXC46. (A) Schematic graph of the pXC46 plasmid. The small squares represent three peaks in the MBR region. Red squares represent the methylated peaks. The triangles represent 12RSS and 23RSS. The arrows represent the relative positions of primers for nested PCR assays. Given that there is NHEJ nucleolytic resection after DSBs occur, a 30-bp zone of interest was delineated at each peak region, designated by two vertical dashed lines. A 30-bp zone of interest is also demarcated for V(D)J recombination breaks at the 12/23RSS coding region. (B) The middle columns of the table show percentages of breakpoints occurring at peak 1, 2, or 3. The right part of the table shows percentages of deletions that have a left side boundary among the MBR peaks and a right side boundary near the 23 coding end (within 30 bp of the 23RSS heptamer). Each data number in this figure is the sum of 4 to 12 independent experiments. The P values connected by solid red lines were calculated using a Student t test comparison between the line 1 values for peak 1 and all of the conditions for peak 1 in lines 2 to 6. The same applies to peak 2 comparisons of line 1 with lines 2 to 6. *, P values connected with the dashed red line represent chi-square test comparisons of methylated peak 1 or peak 2 with the nonmethylated peak 3 when all of the factors are present (line 1). Note that when peaks 1 and 2 are not the focus of breakage, due to omission of one factor (e.g., no CpG methylation, use of mutant AID, or knockout of Artemis), then it is not surprising that the percentage of breaks at all other locations rise, as we see for peak 3 in lines 2, 3, and 5.