The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase
| Autor: | Yuliang Wu, Anthony Kusalik, Oleg Y. Dmitriev, Daniel J. Hogan, Manisha Yadav, Miroslaw Cygler, Shizhuo Yang, Venkatasubramanian Vidhyasagar, Ivy Yeuk Wah Chung, Ravi Shankar Singh |
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| Rok vydání: | 2020 |
| Předmět: |
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 |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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