title: Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5'- to 3'-Helicase Activity with a Preference for Translocation on DNA Rather than RNA.
abstract: In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short noncoding 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 a 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 Escherichia coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but it binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5' to 3' direction, as for the S. pombe protein. When purified from E. coli at a moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the trinucleotide 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.
authors.affiliation: From the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706.
authors.forename: Stephen
authors.initials: S
authors.lastname: Martin-Tumasz
authors.affiliation: From the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 dabrow@wisc.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Brow
mesh_terms: D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D004271:DNA, Fungal; D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C542347:SEN1 protein, S cerevisiae; D004265:DNA Helicases; D020365:RNA Helicases
keywords: DNA/RNA helicase; RNA polymerase II; RNA-binding protein; SF1 helicase; genomic instability; neurodegenerative disease; transcription termination