Functional Role of BLAP75 in BLM-Topoisomerase IIIα-dependent Holliday Junction Processing*S⃞

摘要

The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo)IIIα to form an evolutionarily conserved complex, termed the BTBcomplex, that functions to regulate homologous recombination. BLAP75 bindsDNA, associates with both BLM and Topo IIIα, and enhances the ability ofthe BLM-Topo IIIα pair to branch migrate the Holliday junction (HJ) ordissolve the double Holliday junction (dHJ) structure to yield non-crossoverrecombinants. Here we seek to understand the relevance of the biochemicalattributes of BLAP75 in HJ processing. With the use of a series of BLAP75protein fragments, we show that the evolutionarily conserved N-terminal thirdof BLAP75 mediates complex formation with BLM and Topo IIIα and that theDNA binding activity resides in the C-terminal third of this novel protein.Interestingly, the N-terminal third of BLAP75 is just as adept as thefull-length protein in the promotion of dHJ dissolution and HJ unwinding byBLM-Topo IIIα. Thus, the BLAP75 DNA binding activity is dispensable forthe ability of the BTB complex to process the HJ in vitro. Lastly, weshow that a BLAP75 point mutant (K166A), defective in Topo IIIαinteraction, is unable to promote dHJ dissolution and HJ unwinding by BLM-TopoIIIα. This result provides proof that the functional integrity of theBTB complex is contingent upon the interaction of BLAP75 with TopoIIIα.