Asymmetric processing of human immunodeficiency virus type 1 cDNA in vivo: implications for functional end coupling during the chemical steps of DNA transposition.
Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3' ends of linear transposon or donor DNA are joined to the 5' phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.
Chen
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H
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Engelman
A
A
D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D019895:Transposases / Q000378:metabolism
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D004279:DNA, Viral; D004247:DNA; D019895:Transposases