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.

Highlights

  • Of 200 to 700 amino acids called the helicase core domain, which is formed by two RecA-like domains [2]

  • Using electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), chromatin immunoprecipitation (ChIP)-/ cross-linking immunoprecipitation (CLIP)-Seq, site-directed mutagenesis, and NMR, we found that the KH domain in the DDX43 helicase prefers to bind ssDNA and ssRNA, with a preference for pyrimidine-rich sequences, the tetranucleotide TTGT

  • We showed that the KH domain is crucial for the unwinding processivity of the DDX43 helicase

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Summary

Introduction

Of 200 to 700 amino acids called the helicase core domain, which is formed by two RecA-like domains [2]. Our EMSA and NMR results suggested that the DDX43 KH domain has a nucleic acid sequence binding preference.

Results
Conclusion
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