Holliday junction processing activity of the BLM-Topo IIIalpha-BLAP75 complex.
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 IIIalpha (Topo IIIalpha) and the BLAP75 protein. The BLM-Topo IIIalpha-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 IIIalpha and BLAP75. Enhancement of this BLM activity requires both Topo IIIalpha and BLAP75. Importantly, Topo IIIalpha 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 IIIalpha and BLAP75. However, the topoisomerase activity of Topo IIIalpha 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 IIIalpha.
Bussen
Wendy
W
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Raynard
Steven
S
Busygina
Valeria
V
Singh
Akhilesh K
AK
Sung
Patrick
P
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001816:Bloom Syndrome / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000502:physiology*; D045566:DNA, Cruciform / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D053484:RecQ Helicases; D011995:Recombination, Genetic; D013379:Substrate Specificity
D002352:Carrier Proteins; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C501527:RMI1 protein, human; D006639:Histidine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I