DELTA-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 th at initiates the repair of ultraviolet light induced pyrimidine dimers . Scission of the phosphodiester backbone between the pyrimidines with in a dimer, or 3' to an abasic (AP) site, occurs by a beta-elimination mechanism. In addition, high concentrations of endonuclease V have be en reported to catalyze the cleavage of the C-5-O-P bond in a reaction referred to as delta-elimination. To better understand the enzymology of endonuclease V, the delta-elimination reaction of the enzyme has b een investigated using an oligonucleotide containing a site-specific c is-syn cyclobutane thymine dimer. The slower kinetics of the delta-eli mination reaction compared to beta-elimination and the ability of unla beled dimer-containing DNA to compete more efficiently for delta-elimi nation than beta-elimination indicate that delta-elimination most like ly occurs during a separate enzyme encounter with the incised DNA. Pre vious studies have shown that both the a-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, an d E23D mutants, which are defective in pyrimidine dimer-specific nicki ng, demonstrated that delta-elimination requires Glu-23, but not the p rimary amine at the Fi-terminus. In fact, the T2P mutant was much more efficient at promoting delta-elimination than the wildtype enzyme. Be sides lending further proof that delta-elimination requires a second e ncounter between enzyme and DNA, this result may reflect an enhanced b inding of the T2P mutant to dimer-containing DNA.