DELTA-ELIMINATION BY T4 ENDONUCLEASE-V AT A THYMINE DIMER SITE REQUIRES A SECONDARY BINDING EVENT AND AMINO-ACID GLU-23

被引:26
作者
LATHAM, KA
LLOYD, RS
机构
[1] UNIV TEXAS,MED BRANCH,SEALY CTR MOLEC SCI,DEPT HUMAN BIOL CHEM & GENET,GALVESTON,TX 77555
[2] VANDERBILT UNIV,DEPT BIOCHEM,NASHVILLE,TN 37232
[3] VANDERBILT UNIV,CTR MOLEC TOXICOL,NASHVILLE,TN 37232
关键词
D O I
10.1021/bi00027a031
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 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 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 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, 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 Fi-terminus. In fact, the T2P mutant was much more efficient at promoting delta-elimination than the wildtype 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.
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页码:8796 / 8803
页数:8
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