Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration.
It has long been suspected that a double Holliday junction (dHJ) could be resolved by a topoisomerase partnered with a helicase by convergent branch migration of the HJs. Genetic analysis of yeast TOP3 and SGS1 has lent considerable evidence to the notion that the protein products of these genes are involved in just such a process, although biochemical analysis of the metabolism of a dHJ has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a dHJ substrate that recapitulates many of the features of an endogenous dHJ and represents a much earlier intermediate in the resolution pathway. Here, we show that Drosophila topoisomerase IIIalpha (Topo IIIalpha) and Blm (a homolog of Sgs1) are capable of resolving this substrate to non-cross-over products and that this activity is stimulated by replication protein A (RPA). We investigated the ability of other Drosophila topoisomerases to perform this reaction in concert with Blm and RPA and discovered that this resolution activity is unique to Topo IIIalpha. Examination of the mechanism of resolution reveals that Topo IIIalpha, Blm, and RPA resolve this substrate by convergent migration of the two HJs toward each other, collapsing the dHJ. This mechanism stands in contrast to classic resolvase activities that use a structure-specific endonuclease to cleave the HJs.
Plank
Jody L
JL
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Wu
Jianhong
J
Hsieh
Tao-Shih
TS
D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003434:Crossing Over, Genetic; D004265:DNA Helicases / Q000502:physiology*; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D045566:DNA, Cruciform; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004720:Endonucleases / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000378:metabolism
D003432:Cross-Linking Reagents; D045566:DNA, Cruciform; D029721:Drosophila Proteins; D051716:Replication Protein A; D004720:Endonucleases; D004265:DNA Helicases; C470639:blm protein, Drosophila; D004264:DNA Topoisomerases, Type I