Helicase activity of the Sen1 protein in schizosaccharomyces pombe

Abstract

A DNA and RNA helicase was purified from extracts of Schizosaccharomyces pombe to near homogeneity. The helicase activities copurified with a 95-kDa polypeptide upon a SDS-polyacrylamide gel electrophoresis. Determination of its partial amino acid sequence and molecular masses of its trypsin-digested peptides revealed that the enzyme is homologous to the SEN1 gene product of Saccharomyces cerevisiae, an essential protein affecting a variety of RNA metabolisms in a global fashion including endonuclease cleavage of introns from precursor tRNAs in yeasts. Thus, the purified enzyme was named SpSen1p (Sen1p of Schizosaccharomyces pombe). This study first demonstrated that SpSen1p is indeed a DNA and RNA helicase as inferred from the fact that SEN1 of Saccharomyces cerevisiae shares sequence homology to Upf1, previously known as an RNA and DNA helicase. In addition, various biochemical parameters associated with the enzyme were determined in detail. The helicase utilized partial duplex substrates with free 5``-single-stranded tails, demonstrating that it translocated with the exclusive directionality of a 5`` to 3``. SpSen1p was capable of unwinding all four combinations of DNA and RNA duplexes. In agreement with this observation, SpSen1p hydrolyzed ATP in the presence of either DNA or RNA as cofactor. Among polynucleotide cofactors tested, yeast tRNA was least efficient in supporting ATP hydrolysis of SpSen1p. RNA substrate was unwound more efficiently (2-fold) by SpSen1p than DNA substrate in the absence of salt. However, in the presence of salt (25-50 mM), both DNA and RNA substrates were unwound equally well. The enzyme has higher affinity (5-8 fold) towards single-stranded DNA than single-stranded RNA, whereas RNA was preferred substrates for unwinding. This observation indicates that stronger affinity towards DNA could hinder the enzyme``s ability to translocate along DNA, thereby lowering unwinding efficiency of DNA substrate. Purification of a larger version ...