TY - JOUR
T1 - The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity
AU - Talwar, Tanu
AU - Vidhyasagar, Venkatasubramanian
AU - Qing, Jennifer
AU - Guo, Manhong
AU - Kariem, Ahmad
AU - Lu, Yi
AU - Singh, Ravi Shankar
AU - Lukong, Kiven Erique
AU - Wu, Yuliang
AU - Sung, Patrick
N1 - Funding Information:
This work was supported by the Canadian Breast Cancer Foundation (to Y. W. and K. E. L.). The authors declare that they have no conflicts of interest with the contents of this article.
PY - 2017/6/23
Y1 - 2017/6/23
N2 - The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, also known asHAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. DDX43 is highly expressed in many tumors and is, therefore, considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5′ to 3′ polarity on RNA and a 3′ to 5′ direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full-length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.
AB - The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, also known asHAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. DDX43 is highly expressed in many tumors and is, therefore, considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5′ to 3′ polarity on RNA and a 3′ to 5′ direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full-length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.
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U2 - 10.1074/jbc.M117.774950
DO - 10.1074/jbc.M117.774950
M3 - Article
C2 - 28468824
AN - SCOPUS:85021647705
VL - 292
SP - 10429
EP - 10443
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 25
ER -