1. The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase
- Author
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Yuliang Wu, Anthony Kusalik, Oleg Y. Dmitriev, Daniel J. Hogan, Manisha Yadav, Miroslaw Cygler, Shizhuo Yang, Venkatasubramanian Vidhyasagar, Ivy Yeuk Wah Chung, and Ravi Shankar Singh
- Subjects
0301 basic medicine ,MEME, Multiple Expectation maximization for Motif Elicitation ,substrate specificity ,Biochemistry ,DEAD-box RNA Helicases ,PCBP1, poly(C) binding protein 1 ,DDX43 ,biology ,Chemistry ,Protein Stability ,SELEX ,SELEX Aptamer Technique ,HSQC, heteronuclear single quantum coherence ,RNA Helicase A ,Recombinant Proteins ,3. Good health ,Neoplasm Proteins ,ChIP-seq ,ChIP, chromatin immunoprecipitation ,Ni-NTA, nickel-nitrilotriacetic acid ,Heteronuclear single quantum coherence spectroscopy ,Research Article ,EMSA, electrophoretic mobility shift assays ,CLIP, crosslinking immunoprecipitation ,Protein domain ,DNA, Single-Stranded ,SELEX, systematic evolution of ligands by exponential enrichment ,03 medical and health sciences ,CML, chronic myeloid leukemia ,KH, K-homology ,Humans ,Protein–DNA interaction ,Electrophoretic mobility shift assay ,Molecular Biology ,hnRNP K, heterogeneous nuclear ribonucleoprotein K ,GO, gene ontology ,030102 biochemistry & molecular biology ,CLIP-seq ,HAGE, helicase antigen gene ,DNA Helicases ,Helicase ,Computational Biology ,Cell Biology ,Processivity ,KH domain ,NMR ,030104 developmental biology ,Pyrimidines ,Purines ,biology.protein ,Biophysics ,BSA, bovine serum albumin ,helicase processivity - Abstract
The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation–seq, and cross-linking immunoprecipitation–seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.
- Published
- 2020