The human homolog of Escherichia coli Orn degrades small single-stranded RNA and DNA oligomers.
We report here the identification of human homologues to the essential Escherichia coli Orn protein and the related yeast mitochondrial DNA-escape pathway regulatory factor Ynt20. The human proteins appear to arise from alternatively spliced transcripts, and are thus identical, except the human Ynt20 equivalent contains an NH(2)-terminal extension that possesses a predicted mitochondrial protease cleavage signal. In vitro analysis revealed that the smaller human protein exhibits a 3' to 5' exonuclease activity for small (primarily </=5 nucleotides in length) single-stranded RNA and DNA oligomers. We have named this human protein Sfn for small fragment nuclease to reflect its broad substrate range, and have termed the longer protein hSfnalpha. Sfn prefers Mn(2+) as a metal cofactor and displays a temperature-resistant (to 50 degrees C) nuclease activity. Kinetic analysis indicates that Sfn exhibits a similar affinity for small RNAs and DNAs (K(m) of approximately 1.5 micrometer), but degrades small RNAs approximately 4-fold more efficiently than DNA. Mutation of a conserved aspartate (Asp(136)) to alanine abolishes both nuclease activities of Sfn. Northern blot analysis revealed that a 1-kilobase transcript corresponding to SFN and/or SFNalpha (these mRNAs differ by only two nucleotides) is expressed at varying levels in all fetal and adult human tissues examined. Expressed tag sequence clone analysis found that the two splice variants, SFN to SFNalpha, are present at a ratio of roughly 4 to 1, respectively. The results presented within suggest a role for human Sfn in cellular nucleotide recycling.
Nguyen
L H
LH
Molecular and Structural Biology Division, Lawrence Livermore National Laboratory, Livermore, California 94551, USA.
Erzberger
J P
JP
Root
J
J
Wilson
D M
DM
D048948:14-3-3 Proteins; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D014408:Biomarkers, Tumor; D015152:Blotting, Northern; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases; D020224:Expressed Sequence Tags; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D012313:RNA / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D009363:Neoplasm Proteins; D012313:RNA; D004247:DNA; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human