Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases

被引:22
作者
Skinner, LM
Sudol, M
Harper, AL
Katzman, M
机构
[1] Penn State Univ, Milton S Hershey Med Ctr, Coll Med,Dept Med, Div Infect Dis, Hershey, PA 17033 USA
[2] Penn State Univ, Milton S Hershey Med Ctr, Coll Med, Dept Microbiol & Immunol, Hershey, PA 17033 USA
关键词
D O I
10.1074/jbc.M007032200
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
Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we per formed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immunodeficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.
引用
收藏
页码:114 / 124
页数:11
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