Photolyase-Mediated Repair of (6-4)PP Lesions

Synthesized oligonucleotides with a single (6-4)PP were 3′ end-labeled as described above and purified through G25 gel filtration columns (GE Healthcare). About 0.1 pmol of radiolabeled substrates and 0.5 pmol of the same oligonucleotides unlabeled were incubated with 1.25 pmol of Escherichia coli (6-4)PP photolyase (Selby and Sancar 2012 (link)) in 50 µL of PLR buffer (50 mM Tris-HCl at pH7.5, 100 mM NaCl, 1 mM EDTA, 10 mM DTT) for 15 min or 2 h on ice with the irradiation of 18 mW/cm² 366 nm light from two black-light bulbs (F15T8-BLB, General Electric) filtered through one plate of glass. After phenol-chloroform extraction and ethanol precipitation, the oligonucleotides were incubated with 1 µL of RecJ_f (New England Biolabs) for 1 h at 37°C in 10 µL of 1× NEB2 buffer. Two microliters of reaction mixture was mixed with 10 µL of formamide loading buffer and incubated for 5 min at 95°C before separation with 20% denaturing sequencing gels. The gels were quantified by PhosphorImager (GE Healthcare).