δ-Elimination by T4 Endonuclease V at a Thymine Dimer Site Requires a Secondary Binding Event and Amino Acid Glu-23

Endonuclease V from bacteriophage T4 is a well characterized enzyme that initiates the repair of ultraviolet light induced pyrimidine dimers. Scission of the phosphodiester backbone between the pyrimidines within a dimer, or 3’ to an abasic (AP) site, occurs by a β-elimination mechanism. In addition, high concentrations of endonuclease V have been reported to catalyze the cleavage of the C₅-O-P bond in a reaction referred to as δ-elimination. To better understand the enzymology of endonuclease V, the δ-elimination reaction of the enzyme has been investigated using an oligonucleotide containing a sitespecific cis-syn cyclobutane thymine dimer. The slower kinetics of the δ-elimination reaction compared to β-elimination and the ability of unlabeled dimer-containing DNA to compete more efficiently for δ-elimination than β-elimination indicate that δ-elimination most likely occurs during a separate enzyme encounter with the incised DNA. Previous studies have shown that both the $$-amino group of the N-terminus and the acidic residue Glu-23 are necessary for the N-glycosylase and AP lyase activities of endonuclease V. Experiments with T2P, E23Q, and E23D mutants, which are defective in pyrimidine dimer-specific nicking, demonstrated that δ-elimination requires Glu-23, but not the primary amine at the N-terminus. In fact, the T2P mutant was much more efficient at promoting δ-elimination than the wildtype enzyme. Besides lending further proof that δ-elimination requires a second encounter between enzyme and DNA, this result may reflect an enhanced binding of the T2P mutant to dimer-containing DNA.