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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: DNA Repair (Amst). 2015 May 16;31:80–90. doi: 10.1016/j.dnarep.2015.05.004

Fig. 6.

Fig. 6

mMyh glycosylase activity can be enhanced by 9-1-1 and its subunits. (A) Assay of mMyh glycosylase activity in the presence of human 91-266-1-1, Rad91-266, Rad1, Hus1, and Rad9 (labeled as Rad9full). Lane 1, 5′-32P -labeled A/Go-containing DNA. Lane 2, 0.18 nM A/Go-DNA was incubated with 0.2 nM mMyh. Lanes 3–5 are similar to lane 2 but included 0.4, 1.6, and 6.4 nM 91-266-1-1, respectively. Lane 6, A/Go-DNA was incubated with 6.4 nM 91-266-1-1 without mMyh. Lanes 7–22 are similar to lanes 3–6 except with Rad91-266, Rad1, Hus1, or Rad9full as indicated. The concentrations of Rad91-266, Rad1, Hus1, and Rad9full are based on monomers. Arrows mark the intact DNA substrate (S) and the cleavage product (P). (B) Quantitative analyses of mMyh glycosylase stimulation by 91-266-1-1 and its subunits from results in (A). Fold stimulation was calculated as in Fig. 4B. Error bars indicate SD; n = 3. (C) Rad9 lacking the C-terminal domain has higher affinity for MYH than does intact Rad9. Immobilized GST, GST-hRad9full, and GST-hRad91-266 constructs were used to pull-down His-mMyh. GST pull-down experiments were performed similar to Fig. 3C.