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. 2013 Nov 11;110(51):20437–20442. doi: 10.1073/pnas.1314289110

Fig. 4.

Fig. 4.

Splicing of DNA 3′-PO4 and 5′-OH ends by RtcB. (A) Reaction mixtures (10 µL) containing 50 mM Tris⋅HCl (pH 8.0), 2 mM MnCl2, 200 µM GTP, 1 pmol (0.1 µM) 5′ 32P-labeled pD12p substrate, 50 pmol (5 µM) of a complementary HODNA acceptor strand (HOGATATCGCAATTG), and RtcB as specified were incubated at 37 °C for 20 min. The products were analyzed by urea-PAGE and visualized by autoradiography. (B) Schematic diagram of the pathway of DNA splicing by RtcB. (C) Reaction mixtures (10 µL) containing 50 mM Tris⋅HCl (pH 8.0), 2 mM MnCl2, 200 µM GTP, 1 pmol (0.1 µM) 5′ 32P-labeled pD12p, 0 or 100 pmol (10 µM) RtcB, and 100 pmol (10 µM) of complementary (HODNAc) or noncomplementary (HODNAnc) DNA acceptors strand as indicated were incubated at 37 °C for 20 min. The products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the pD12p substrate, pD12ppG intermediate, and spliced product are indicated (Right).