Extended Data Figure 4: Sae2 does not show nuclease activity, and does not promote MRX nuclease on hairpins. | Nature

Extended Data Figure 4: Sae2 does not show nuclease activity, and does not promote MRX nuclease on hairpins.

From: Sae2 promotes dsDNA endonuclease activity within Mre11–Rad50–Xrs2 to resect DNA breaks

Extended Data Figure 4

a, Recombinant Sae2 was assayed on a dsDNA substrate in the presence of either magnesium (5 mM) or manganese (5 mM), with or without RPA (23 nM), as indicated. Free label, carryover of [32P]ATP from the labelling reaction, marks the position of the smallest possible product resulting from potential nuclease activity. Samples were analysed on a 10% native polyacrylamide gel. b, Recombinant Sae2 was assayed on a Y-structure DNA substrate either in the presence of magnesium (5 mM) or manganese (5 mM), with or without RPA (23 nM), as indicated. Free label, carryover of [32P]ATP from the labelling reaction, marks the position of the smallest possible product resulting from potential nuclease activity. Samples were analysed on a 10% native polyacrylamide gel. c, Recombinant Sae2 was assayed on a HL-3 hairpin DNA substrate either in the presence of magnesium (5 mM) or manganese (5 mM), with or without RPA (23 nM), as indicated. Samples were analysed on a 15% denaturing polyacrylamide gel. d, Nuclease assay was performed with MRX and Sae2 on HP-2 DNA, as indicated. Samples were analysed on a 15% denaturing polyacrylamide gel. Sae2 does not promote endonuclease of MRX on HP-2 DNA substrate. e, Nuclease assay was performed with MRX and Sae2 on HL-3 hairpin DNA, as indicated. Samples were analysed on a 15% denaturing polyacrylamide gel. Sae2 does not promote endonuclease of MRX on HL-3 DNA substrate.

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