The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase

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 single-stranded (ss)DNA and ssRNA. Electrophoretic mobility shift assay (EMSA) and nuclear magnetic resonance (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. SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, and crosslinking immunoprecipitation (CLIP)-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-HSQC 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 strand 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.