Purification and enzymatic characterization of Gallus gallus BLM helicase.
Mutations in human BLM helicase give rise to the autosomal recessive Bloom syndrome, which shows high predisposition to types of malignant tumours. Though lots of biochemical and structural investigations have shed lights on the helicase core, structural investigations of the whole BLM protein are still limited due to its low stability and production. Here by comparing with the expression systems and functions of other BLM homologues, we developed the heterologous high-level expression and high-yield purification systems for Gallus gallus BLM (gBLM) in Escherichia coli. Subsequent DNA binding and unwinding determinations demonstrated that gBLM was a vigorous atypical DNA structure specific helicase, which not only showed high preference for the 3'-tailed DNA structures but also could efficiently unwind bubble DNA structures with blunt-ends, indicating its biological roles in processing DNA metabolism intermediates. Further comparative analysis between gBLM and gBLM Core revealed that the long N-terminal domain facilitated the binding affinity of forked and bubble DNA structures and it was also required for the DNA unwinding activities of gBLM. Thus, we present the first enzymatic characterization of gBLM and its N-terminal domain, providing a new model for probing the mechanism and structure of human BLM.
College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.
Jing
J
Shi
College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.
Na-Nv
NN
Liu
College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.
Yan-Tao
YT
Yang
College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.|Laboratoire de Biologie et Pharmacologie Appliquée, ENS de Cachan, Université Paris-Saclay, CNRS, 61 Avenue du Présidnt Wilson, Cachan 94235, France.
Xu-Guang
XG
Xi
D000818:Animals; D002645:Chickens; D053484:RecQ Helicases / Q000302:isolation & purification* / Q000378:metabolism*
C097151:Bloom syndrome protein; D053484:RecQ Helicases
BLM helicase; Bloom syndrome; DNA binding; DNA unwinding; N-terminal domain