Recombinogenic flap ligation mediated by human topoisomerase I.
Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.
Andersen
Félicie F
FF
Department of Molecular Biology, University of Aarhus, C.F. Møllers Allé, Building 130, DK-8000, C, Aarhus, Denmark.
Andersen
Kirsten E
KE
Kusk
Mette
M
Frøhlich
Rikke F
RF
Westergaard
Ole
O
Andersen
Anni H
AH
Knudsen
Birgitta R
BR
D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D017384:Sequence Deletion; D013379:Substrate Specificity
D008024:Ligands; D011994:Recombinant Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I