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

Lynn M. Skinner, Malgorzata Sudol, Amy L. Harper, Michael Katzman

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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 performed 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.

Original languageEnglish (US)
Pages (from-to)114-124
Number of pages11
JournalJournal of Biological Chemistry
Volume276
Issue number1
DOIs
StatePublished - Jan 5 2001

Fingerprint

Integrases
Nucleophiles
Endonucleases
Human Activities
Sheep
DNA
Alcohols
Viruses
Processing
Visna-maedi virus
Human Immunodeficiency Virus Proteins
Rous sarcoma virus
Water
Disintegration
Viral DNA
Enzymes
Catalysis
Joining
Conformations
HIV-1

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "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 performed 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.",
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Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases. / Skinner, Lynn M.; Sudol, Malgorzata; Harper, Amy L.; Katzman, Michael.

In: Journal of Biological Chemistry, Vol. 276, No. 1, 05.01.2001, p. 114-124.

Research output: Contribution to journalArticle

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