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 Esch erichia coli Orn protein and the related yeast mitochondrial DNA-escape pat hway regulatory factor Ynt20, The human proteins appear to arise from alter natively spliced transcripts, and are thus identical, except the human Ynt2 0 equivalent contains an NH2-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 less than or equal to 5 nucleotides in length) single-stranded RN A and DNA oligomers, We have named this human protein Sfn for Small fragmen t nuclease to reflect its broad substrate range, and have termed the longer protein hSfn alpha. Sfn prefers Mn2+ as a metal cofactor and displays a te mperature-resistant (to 50 degrees C) nuclease activity. Kinetic analysis i ndicates that Sfn exhibits a similar affinity for small RNAs and DNAs (K-m of similar to 1.5 mu M), but degrades small RNAs similar to 4-fold more eff iciently than DNA, Mutation of a conserved aspartate (Asp(136)) to alanine abolishes both nuclease activities of Sfn, Northern blot analysis revealed that a l-kilobase transcript corresponding to SFN and/or SFN alpha (these m RNAs differ by only two nucleotides) is expressed at varying levels in all fetal and adult human tissues examined. Expressed tag sequence clone analys is found that the two splice variants, SFN to SFN alpha, are present at a r atio of roughly 4 to 1, respectively. The results presented within suggest a role for human Sfn in cellular nucleotide recycling.