title: Purified human SUV3p exhibits multiple-substrate unwinding activity upon conformational change.
abstract: Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, the helicase domain in both yeast and human SUV3 varies considerably from the typical RNA helicase motifs. To investigate its enzymatic activities, a homogeneously purified preparation of SUV3 is required. Expression of a processed form of human SUV3 carrying an N-terminal deletion of 46 amino acids (SUV3DeltaN46) in a yeast suv3 null mutant, which otherwise fails to grow in a nonfermentable carbon source and forms petite colonies in glucose medium, rescues the null phenotype. Through a five-step chromatographic procedure, an 83 kDa SUV3DeltaN46 protein (SUV3-83) and a partially degraded 70 kDa product (SUV3-70) containing amino acids 68-685 were purified to homogeneity. Single- or double-stranded DNA and RNA stimulated ATPase activity of both proteins. SUV3-70, which retains core catalytic domains, can bind and unwind multiple duplex substrates of RNA and DNA with a 5'-3' directionality over a wide range of pH, while SUV3-83 has helicase activity at only acidic pH. ATP, but not nonhydrolyzable ATP, is essential for the unwinding activity, suggesting the requirement of the energy derived from ATP hydrolysis. Consistent with this notion, suv3 mutants containing alanine (A) or arginine (R) substitutions at the conserved lysine residue in the ATP binding site (K213) lost ATPase activity and also failed to unwind the substrates. Importantly, circular dichroism (CD) spectral analysis showed that SUV3-83, at pH 5.0, adopts a conformation similar to that of SUV3-70, suggesting a conformational change in SUV3-83 is required for its helicase activity. The physiological relevance of the multiple-substrate helicase activity of human SUV3 is discussed.
authors.affiliation: Department of Biological Chemistry, College of Medicine, University of California, Irvine, California 92697, USA.
authors.forename: Zhanyong
authors.initials: Z
authors.lastname: Shu
authors.forename: Sangeetha
authors.initials: S
authors.lastname: Vijayakumar
authors.forename: Chi-Fen
authors.initials: CF
authors.lastname: Chen
authors.forename: Phang-Lang
authors.initials: PL
authors.lastname: Chen
authors.forename: Wen-Hwa
authors.initials: WH
authors.lastname: Lee
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D020816:Amino Acid Motifs; D053487:DEAD-box RNA Helicases; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D020365:RNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D009692:Nucleic Acid Heteroduplexes; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C469673:SUPV3L1 protein, human; C075918:SUV3 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases