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. 2013 Jul 1;41(17):8182–8195. doi: 10.1093/nar/gkt588

Figure 7.

Figure 7.

Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. (A) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. (B) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. (C) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. (D) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. (E) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. (F) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.