S. cerevisiae Sen1 Helicase Domain Exhibits 5′ to 3′ Helicase Activity With a Preference for Translocation on DNA Rather Than RNA [RNA]

July 21st, 2015 by Martin-Tumasz, S., Brow, D. A.

In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short non-coding RNA genes by RNA polymerase II. However, the mechanism by which Sen1 promotes transcription termination is not known. Prior biochemical studies on the Sen1 homolog from Schizosaccharomyces pombe showed that it can bind and unwind both DNA and RNA, but the S. pombe protein is not essential and has not been demonstrated to function in transcription. Furthermore, Sen1 from either yeast has not previously been expressed as recombinant protein, due to its large molecular mass (252 kDa in S. cerevisiae). Here we report the purification and characterization of the 89 kDa S. cerevisiae Sen1 helicase domain (Sen1-HD) produced in E. coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5ʹ to 3ʹ direction, as for the S. pombe protein. When purified from E. coli at moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the tri-nucleotide repeat (CAN)4. We propose that Sen1 binds to RNAs and prevents their stable pairing with DNA, consistent with in vivo studies by others showing increased R-loop (RNA/DNA hybrid) formation when Sen1 activity is impaired by mutations. Our results are consistent with a model in which Sen1 promotes transcription termination by resolving R-loops.