Functional role of BLAP75 in BLM-topoisomerase IIIalpha-dependent holliday junction processing.
The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo) IIIalpha 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 IIIalpha, and enhances the ability of the BLM-Topo IIIalpha 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 IIIalpha 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 IIIalpha. 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 IIIalpha interaction, is unable to promote dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. This result provides proof that the functional integrity of the BTB complex is contingent upon the interaction of BLAP75 with Topo IIIalpha.
Raynard
Steven
S
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Zhao
Weixing
W
Bussen
Wendy
W
Lu
Lucy
L
Ding
Yang-Yang
YY
Busygina
Valeria
V
Meetei
Amom Ruhikanta
AR
Sung
Patrick
P
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D053484:RecQ Helicases
D002352:Carrier Proteins; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; C501527:RMI1 protein, human; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I