The DNase activity of RNase T and its application to DNA cloning

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escher ichia coli, The enzyme plays an important role in stable RNA metabolism, in cluding tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-strand ed (ds) stem. In the course of study of its specificity and mechanism, we f ound that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA muc h move tightly and shows less sequence specificity. As with RNA, DNA second ary structure strongly affects its degradation by RNase T, Thus, RNase T ac tion on a dsDNA with a single-stranded 3'-extension efficiently generates b lunt-ended DNA. This property of RNase T suggested that it might be a usefu l enzyme for blunt-ended DNA cloning. We show here that RNase T provides mu ch higher cloning efficiency than the currently used mung bean nuclease.