5'-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5'-exonuclease-1.
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.
Stevens
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Biology Division, Oak Ridge National Laboratory, Tennessee 37831-8080, USA.
Poole
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D000595:Amino Acid Sequence; D003851:Deoxyribonucleases / Q000378:metabolism; D005095:Exoribonucleases; D005656:Fungal Proteins / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D013379:Substrate Specificity
D005656:Fungal Proteins; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; C075577:RAT1 protein, S cerevisiae; D011061:Poly A; D012343:RNA, Transfer; D003851:Deoxyribonucleases; D005095:Exoribonucleases; D012260:Ribonucleases; C068398:XRN1 protein, S cerevisiae; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I