Extended Data Fig. 2: DNA cleavage optimization.
From: Reconstitution of SPO11-dependent double-strand break formation
In all panels, representative gels are above, quantification is below (mean ± s.d. of n = 3 experiments). a, Metal ion dependence of DNA cleavage. SPO11–TOP6BL complexes (100 nM) were incubated with 4 ng/µl pUC19 DNA in the presence of the indicated concentration of MnCl2, MgCl2, or CaCl2. b, Temperature dependence. Reactions contained 100 nM SPO11 complexes and 4 ng/µl pUC19 DNA with 1 mM MnCl2. c, pH dependence. Reactions contained 100 nM SPO11 complexes and 4 ng/µl pUC19 DNA with 1 mM MnCl2. The pH 9.0 and pH 10.0 conditions resulted in a high background of SPO11-independent nicking (right lanes), so these samples were omitted from the quantification. d, Comparison of positively and negatively supercoiled substrates. Reactions contained 100 nM SPO11 complexes and 4 ng/µl pUC19 DNA with 1 mM MnCl2. e, Comparison of relaxed covalently closed circle (CCC) and negatively supercoiled substrates. Reactions contained 100 nM SPO11 complexes and 4 ng/µl pUC19 DNA with 1 mM MnCl2. For this experiment, reaction products were separated on agarose gels containing ethidium bromide. f, Cleavage of a linear DNA substrate. Reactions contained 100 nM SPO11 complexes and 4 ng/µl of a linear DNA fragment from pUC19 (mix of cold and 5′ 32P-labeled on both ends) with 5 mM MnCl2. Deproteinized reaction products were divided and aliquots were run separately on native PAGE to detect DSBs (left) and denaturing urea PAGE to detect both nicks and DSBs (right).
