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J. Biol. Chem., Vol. 283, Issue 23, 15701-15708, June 6, 2008

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Functional Role of BLAP75 in BLM-Topoisomerase III-dependent Holliday Junction Processing^*

Steven Raynard, Weixing Zhao, Wendy Bussen¹, Lucy Lu, Yang-Yang Ding, Valeria Busygina, Amom Ruhikanta Meetei, and Patrick Sung²

From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520 and the Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229

The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo) III to form an evolutionarily conserved complex, termed the BTB complex, that functions to regulate homologous recombination. BLAP75 binds DNA, associates with both BLM and Topo III, and enhances the ability of the BLM-Topo III pair to branch migrate the Holliday junction (HJ) or dissolve the double Holliday junction (dHJ) structure to yield non-crossover recombinants. Here we seek to understand the relevance of the biochemical attributes of BLAP75 in HJ processing. With the use of a series of BLAP75 protein fragments, we show that the evolutionarily conserved N-terminal third of BLAP75 mediates complex formation with BLM and Topo III and that the DNA binding activity resides in the C-terminal third of this novel protein. Interestingly, the N-terminal third of BLAP75 is just as adept as the full-length protein in the promotion of dHJ dissolution and HJ unwinding by BLM-Topo III. Thus, the BLAP75 DNA binding activity is dispensable for the ability of the BTB complex to process the HJ in vitro. Lastly, we show that a BLAP75 point mutant (K166A), defective in Topo III interaction, is unable to promote dHJ dissolution and HJ unwinding by BLM-Topo III. This result provides proof that the functional integrity of the BTB complex is contingent upon the interaction of BLAP75 with Topo III.

Received for publication, March 17, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Research Grants ES015252 and ES015632 (to P. S.) and R01 HL084082 (to A. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Present address: Dept. of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, St. Louis, MO 63108.

² To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University School of Medicine, C130 Sterling Hall of Medicine, 333 Cedar St., New Haven, CT 06520-8024. Tel.: 203-785-4553; Fax: 203-785-6404; E-mail: patrick.sung{at}yale.edu.

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