Publications

• 5'-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5'-exonuclease-1.

  Stevens A., Poole T.L.

  5'-Exonuclease-2 has been purified 17,000-fold from whole cell extracts of Saccharomyces cerevisiae. A 116-kDa polypeptide parallels the enzyme activity when the purified protein is examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Amino-terminal sequencing of the 116-kDa p ... >> More

  5'-Exonuclease-2 has been purified 17,000-fold from whole cell extracts of Saccharomyces cerevisiae. A 116-kDa polypeptide parallels the enzyme activity when the purified protein is examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Amino-terminal sequencing of the 116-kDa protein shows that the sequence agrees with that encoded by the HKE1 gene, previously reported to encode exonuclease-2. A 45-kDa polypeptide also parallels the enzyme activity upon purification, and Sephacryl S-200 molecular sieve chromatography of the purified enzyme shows a parallel elution of most of the 116- and 45-kDa polypeptides, suggesting a close association of the two. Enzyme instability has precluded a more detailed analysis of their associative properties. The enzyme hydrolyzes RNA substrates to 5'-mononucleotides in a processive manner. Measurements of its substrate specificity and mode of action are compared with 5'-exonuclease-1. Restriction cut single-stranded T7 DNA is hydrolyzed at approximately 5-7% of the rate of 18 S rRNA of yeast by both enzymes. That 5'-exonuclease-2 hydrolyzes in a processive manner and lacks endonuclease activity is shown by the finding that [5'-32P]GMP is the only product of its hydrolysis of [alpha-32P]GTP-labeled synthetic RNAs. That 5'-exonuclease-2 hydrolyzes by a 5'-->3' mode is shown by: 1) its poor hydrolysis of both 5'-capped and triphosphate-ended RNA substrates; 2) the products of its hydrolysis of [5'-32P,3H](pA)4; and 3) the accumulation of 3'-stall fragments when a strong artificial RNA secondary structure is present in synthetic RNAs. 5'-Exonuclease-1 hydrolyzes the synthetic RNAs and (pA)4 in an identical manner. << Less

  J. Biol. Chem. 270:16063-16069(1995) [PubMed] [EuropePMC]

• A 5'----3' exoribonuclease of human placental nuclei: purification and substrate specificity.

  Stevens A., Maupin M.K.

  An exoribonuclease that hydrolyzes single-stranded RNA by a 5'----3' mode yielding 5'-mononucleotides has been purified from human placental nuclei. Chromatographic studies of crude placental nuclear extracts suggest that the enzyme is a relatively abundant nuclear RNase. Poly(A) is degraded by a ... >> More

  An exoribonuclease that hydrolyzes single-stranded RNA by a 5'----3' mode yielding 5'-mononucleotides has been purified from human placental nuclei. Chromatographic studies of crude placental nuclear extracts suggest that the enzyme is a relatively abundant nuclear RNase. Poly(A) is degraded by a processive mechanism while rRNA is degraded in a partially non-processive manner, possibly because of its secondary structure. The enzyme has an apparent molecular weight of 113,000, derived from determinations of the Stokes radius (43 A) and sedimentation coefficient (6.3 S). Substrates with 5'-phosphomonoester end groups are 10-20 times better than 5'-dephosphorylated substrates. The locale of the enzyme in nuclei of normal human cells as well as its mode of action suggest a role in nuclear RNA processing or turnover. << Less

  Nucleic Acids Res 15:695-708(1987) [PubMed] [EuropePMC]

• An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity.

  Kenna M., Stevens A., McCammon M., Douglas M.G.

  An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as w ... >> More

  An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as well as activities involving chromosomal DNA pairing and mechanics. Preliminary analysis of an hke1ts mutant reveals a precipitous decline in the translation of mRNA at the nonpermissive temperature. Sporulation of heterozygous HKE1/hke1::URA3 diploids reveals that this gene, unlike the highly related XRN1/KEM1 gene, is essential for cell viability. Overexpression of the homologous gene product, p175, failed to rescue cells lacking a functional p116. In vitro studies demonstrate that p116 is a protein with 5'-->3' exoribonuclease activity, a major activity of the related p175. An immunoreactive RNase activity of 116 kDa is abolished with antiserum against p116. Both the level of this protein and the RNase activity correlate with HKE1 gene dosage. The RNase activity purifies coincidentally with a previously described 116-kDa RNase having 5'-->3' exoribonuclease activity. << Less

  Mol. Cell. Biol. 13:341-350(1993) [PubMed] [EuropePMC]

• Purification and characterization of a Saccharomyces cerevisiae exoribonuclease which yields 5'-mononucleotides by a 5' leads to 3' mode of hydrolysis.

  Stevens A.

  An exoribonuclease producing 5'-mononucleotides has been purified from ribosomes of Saccharomyces cerevisiae. The enzyme has a broad pH optimum around 8.0, requires divalent cation, and is stimulated by monovalent cation with the cation and degree of stimulation being dependent on the substrate us ... >> More

  An exoribonuclease producing 5'-mononucleotides has been purified from ribosomes of Saccharomyces cerevisiae. The enzyme has a broad pH optimum around 8.0, requires divalent cation, and is stimulated by monovalent cation with the cation and degree of stimulation being dependent on the substrate used. With either poly(A) or rRNA as substrate, the enzyme has a processive mode of hydrolysis. The oligonucleotides, (pA)3-5, are hydrolyzed by the enzyme, and the hydrolysis is dependent on a 5'-phosphate end group. Phosphorylation of the 3' end has little effect on the rate of hydrolysis. With [3H]poly(A) or [3H]rRNA, labeled differentially at the 5' termini, a more rapid release of 5'-terminal label can be shown, providing evidence that the enzyme hydrolyzes in a 5' leads to 3' direction. Further evidence for a 5' leads to 3' mode of hydrolysis is provided by a study of the products of the hydrolysis of [3H](pA)5 labeled at the 5' termini with 32P. No 32P label is found in (pA)2 which accumulates as an intermediate. << Less

  J Biol Chem 255:3080-3085(1980) [PubMed] [EuropePMC]

• A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity.

  Stevens A., Maupin M.K.

  The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate- ... >> More

  The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA). << Less

  Arch Biochem Biophys 252:339-347(1987) [PubMed] [EuropePMC]