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Volume 270, Number 27, Issue of July 07, pp. 16063-16069, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Audrey Stevens , Toni L. Poole

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`-P]GMP is the only product of its hydrolysis of [-P]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`-P,H](pA); 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) in an identical manner.

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