CHARACTERIZATION OF A NOVEL CIS-SYN AND TRANS-SYN-II PYRIMIDINE DIMERGLYCOSYLASE AP LYASE FROM A EUKARYOTIC ALGAL VIRUS, PARAMECIUM-BURSARIA CHLORELLA VIRUS-1

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-sp ecific glycosylase, Recently, the first sequence homolog of T4 endonuc lease V was identified from chlorella virus Paramecium bursaria chlore lla virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interest ingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimid ine dimer, but also for the trans-syn-II isomer, This is the first tra ns-syn-II-specific glycosylase identified to date. Kinetic analysis de monstrates that DNAs containing both types of pyrimidine dimers are cl eaved by the enzyme with similar catalytic efficiencies. Cleavage anal ysis and covalent trapping experiments demonstrate that the enzyme mec hanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino inter mediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics whic h are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic at traction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possi bility that there may be other T4 endonuclease V-like enzymes with spe cificity toward other DNA photoproducts.