Holliday junction processing activity of the BLM-TopoIIIalpha -BLAP75 complex

doi:10.1074/jbc.M706116200

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Papers In Press, published online ahead of print August 28, 2007
J. Biol. Chem, 10.1074/jbc.M706116200
Submitted on July 25, 2007
Revised on August 27, 2007
Accepted on August 28, 2007

Holliday junction processing activity of the BLM-TopoIII $alpha$ -BLAP75 complex

Wendy Bussen, Steven Raynard, Valeria Busygina, Akhilesh K. Singh, and Patrick Sung

Dept of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520

Corresponding Author: patrick.sung{at}yale.edu

BLM, the protein mutated in Bloom’s syndrome, possesses a helicase activity that can dissociate DNA structures, including the Holliday junction, expected to arise during homologous recombination. BLM is stably associated with topoisomerase III $alpha$ (Topo III $alpha$ ) and the BLAP75 protein. The BLM-Topo III $alpha$ -BLAP75 (BTB) complex can efficiently resolve a DNA substrate that harbors two Holliday junctions (the double Holliday junction) in a non-crossover manner. Here we show that the Holliday junction unwinding activity of BLM is greatly enhanced as a result of its association with Topo III $alpha$ and BLAP75. Enhancement of this BLM activity requires both Topo III $alpha$ and BLAP75. Importantly, Topo III $alpha$ cannot be substituted by Escherichia coli Top3, and the Holliday junction unwinding activity of BLM-related helicases WRN and RecQ is likewise impervious to Topo III $alpha$ and BLAP75. However, the topoisomerase activity of Topo III $alpha$ is dispensable for the enhancement of the DNA unwinding reaction. We have also ascertained the requirement for the BLM ATPase activity in double Holliday junction dissolution and DNA unwinding by constructing, purifying, and characterizing specific mutant variants that lack this activity. These results provide valuable information concerning how the functional integrity of the BTB complex is governed by specific protein-protein interactions among the components of this complex and the enzymatic activities of BLM and Topo III $alpha$ .

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Holliday junction processing activity of the BLM-TopoIII-BLAP75 complex

Holliday junction processing activity of the BLM-TopoIII $alpha$ -BLAP75 complex