Functional characteristics of a highly specific integrase encoded by an LTR-retrotransposon.
BACKGROUND
The retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 5'-CGCGCg-3' consensus-sequences. To enlighten the determinants of this high integration specificity, we investigated the functional properties of its integrase protein denoted ZAM-IN.


RESULTS
Here we show that ZAM-IN displays the property to nick DNA molecules in vitro. This endonuclease activity targets specific sequences that are present in a 388 bp fragment taken from the white locus and known to be a genomic ZAM integration site in vivo. Furthermore, ZAM-IN displays the unusual property to directly bind specific genomic DNA sequences. Two specific and independent sites are recognized within the 388 bp fragment of the white locus: the CGCGCg sequence and a closely apposed site different in sequence.


CONCLUSIONS
This study strongly argues that the intrinsic properties of ZAM-IN, ie its binding properties and its endonuclease activity, play an important part in ZAM integration specificity. Its ability to select two binding sites and to nick the DNA molecule reminds the strategy used by some site-specific recombination enzymes and forms the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.
Faye
Babacar
B
UMR/CNRS 6247, Clermont Université, INSERM, U931, Faculté de Médecine, Clermont-Ferrand, France.
Arnaud
Frederick
F
Peyretaillade
Eric
E
Brasset
Emilie
E
Dastugue
Bernard
B
Vaury
Chantal
C
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D016679:Genome, Viral; D005982:Glutathione Transferase / Q000378:metabolism; D019426:Integrases / Q000378:metabolism*; D008957:Models, Genetic; D016296:Mutagenesis; D011995:Recombination, Genetic; D018626:Retroelements / Q000235:genetics*; D020079:Terminal Repeat Sequences; D016662:Virus Integration
D018626:Retroelements; D004247:DNA; D005982:Glutathione Transferase; D019426:Integrases; D004720:Endonucleases
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