Abstract
Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate 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 which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.
Highlights
DNA damage caused by ultraviolet (UV) light is of great interest because it may lead to mutations, carcinogenesis, and cell death
The trans-syn activities are greater than T4 endonuclease V in these assays, with the cv-PDG nicking at trans-syn-I and trans-syn-II dimers 30 –70 times more efficiently than T4 endonuclease V
The cv-PDG cleavage product at ϳ20 nucleotides is consistent with a mechanism similar to T4 endonuclease V, in which the labeled product has a 3Ј-␣,unsaturated aldehyde resulting from a -elimination reaction at the abasic site produced by the initial glycosidic bond cleavage at the pyrimidine dimer
Summary
Polymerase Chain Reaction Cloning and Overexpression of the PBCV-1 pdg Gene—The entire pdg gene (open reading frame A50L) of 426 bp was amplified from PBCV-1 genomic DNA using the polymerase chain reaction. All oligonucleotides containing the photoproducts, other modifications, or control sequence were ␥-32P-labeled ([␥-32P]ATP, 3000 Ci/mmol, NEN Life Science Products) on the 5Ј-end with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) following standard procedures and annealed to their complementary strands to form duplex DNA. Enzyme Activity Assays—Single-stranded or duplex DNAs (0.5–1 nM) containing the modifications were incubated with the indicated amounts of cv-PDG or T4 endonuclease V in the standard reaction buffer containing 25 mM sodium phosphate (pH 6.8), 100 mM NaCl, 1 mM EDTA, and 100 g/ml BSA for 30 min at 37 °C. An equal volume of loading buffer (95% (v/v) formamide, 20 mM EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue) was added to the reactions and the samples heated (except for AP-containing DNA) to 90 °C for 2 min prior to loading onto a 15% denaturing polyacrylamide gel (8 M urea). 1 M piperidine was added to the reaction and incubated for 30 min at 90 °C prior to the addition of gel loading buffer
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