Using RecA and an oligonucleotide to form a stable triple helix
The following is performed as the functional assay for RecA. A triple helix is used to block methylation at one of five HpyCH4IV sites on pUC19. The same ratio of RecA and oligo can be used to set up stable triple helixes in the presence of 0.3 mM ATP γ-S for other applications.
The plasmid pUC19 contains 5 HpyCH4IV sites. A 60 mer was designed with complementarity to the region centered around the HpyCH4IV site at position 374. A reaction containing 1 μg pUC19, 0.18 μg 60 mer, 0.3 mM ATP γ-S, 4 μg RecA, in 40 μl 1X RecA Reaction Buffer was incubated at 37°C for 10 minutes to form a stable triple helix. The unprotected sites were methylated using 8 units of SssI supplemented with160 μM SAM for 10 minutes at 37°C. The reaction was stopped and the triple helix was disrupted by incubation at 65°C for 15 minutes. The reaction was cooled and 10 units of HpyCH4IV were added followed by digestion at 37°C for 20 minutes. > 90% of the product is single cut pUC19.