Table 1. Biochemically characterized DDX5/Dbp2 mutants.

The biochemical properties of DEAD-box proteins are not necessarily coupled. We observe that the literature sometimes uses “helicase-dead” mutants to demonstrate that the RNA helicase activities are not required for certain biological functions of the DDX5/Dbp2 subfamily. However, while such statements are true, these mutants may still be partially active. ATP hydrolysis is required for recycling DEAD-box proteins, allowing multiple rounds of duplex separation, but is not necessary for RNA-binding, or a single round of strand separation (Liu et al. 2008). Therefore, a mutant lacking ATP hydrolysis may still possess RNA-binding activity. For example, DDX5-D248N, a frequently used “enzymatically-dead” mutant that harbors a point mutation in motif II (Walker B motif), possesses equal RNA binding activity and reduced ATPase activity as compared to wild type DDX5. This suggests that the DDX5-D248N mutant can act as an “RNA clamp” that binds to RNA but does not readily dissociate. Taking the ensemble of biochemical properties into account is necessary to decipher the roles of DEAD-box proteins in biological processes. n.d. stands for not determined.

Mutants	Motif	ATP binding	ATP hydrolysis	RNA binding	RNA unwinding	RNA annealing	References
DDX5-K144N	I	None	None	Reduced	None	n.d.	Zhang et al., 2016, Jalal et al., 2007
DDX5-D248N	II	n.d.	Partial	Wild-type	n.d.	n.d.	Zhang et al., 2016
DDX5-E249Q	II	Wild-type	None	n.d.	None	n.d.	Jalal et al., 2007
DDX5-R403L	V	Wild-type	None	n.d.	None	n.d.	Lin et al., 2005
DDX5-R428L	VI	Wild-type	None	n.d.	None	n.d.	Lin et al., 2005
Dbp2-K163N	I	n.a.	None	n.a.	n.a.	n.a.	Cloutier et al., 2012
Dbp2-D268Q	II	n.a.	None	n.a.	n.a.	n.a.	Cloutier et al., 2012