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 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 beta-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 delta-elimination. To better understand the enzymology of endonuclease V, the delta-elimination reaction of the enzyme has been investigated using an oligonucleotide containing a site-specific cis-syn cyclobutane thymine dimer. The slower kinetics of the delta-elimination reaction compared to beta-elimination and the ability of unlabeled dimer-containing DNA to compete more efficiently for delta-elimination than beta-elimination indicate that delta-elimination most likely occurs during a separate enzyme encounter with the incised DNA. Previous studies have shown that both the alpha-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 delta-elimination requires Glu-23, but not the primary amine at the N-terminus. In fact, the T2P mutant was much more efficient at promoting delta-elimination than the wild-type enzyme. Besides lending further proof that delta-elimination requires a second encounter between enzyme and DNA, this result may reflect an enhanced binding of the T2P mutant to dimer-containing DNA.
Latham
K A
KA
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA.
Lloyd
R S
RS
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D014764:Viral Proteins; D005973:Glutamine; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)