Identification of a Potent DNase Activity Associated with RNase T of Escherichia coli

Mohan Viswanathan,Ken W Dower,Susan T Lovett

doi:10.1074/jbc.273.52.35126

Abstract

RNase T was first identified as an enzyme responsible for end turnover of tRNA in Escherichia coli. Its activity, specific for tRNA-C-C-A, catalyzes the release of tRNA-C-C and AMP. RNase T, along with several other RNases, plays a role in maturation of several other RNA species by a similar limited nuclease activity. In previous work, we identified the gene for RNase T, rnt, as a high copy suppressor of the UV sensitivity conferred by deficiency in three single-strand DNA-specific exonucleases, RecJ, exonuclease I, and exonuclease VII. This suggested that RNase T may process DNA substrates as well. In this work, we show that purified RNase T possesses a potent 3' to 5' single-strand DNA-specific exonucleolytic activity. Its Km for single-strand DNA substrates is many orders of magnitude lower than that for tRNA, suggesting that single-strand DNA may be a natural biological substrate for RNase T. We suggest that the DNase activity of RNase T may play a role in end trimming reactions during DNA recombination and/or DNA repair.

Highlights

RNase T of Escherichia coli was identified as a 3Ј exoribonuclease due to its ability to remove the terminal adenine residue from uncharged tRNA-C-C-A molecules [1]
Overexpression and Purification of a DNase Activity Associated with RNase T—The plasmid pAL1 containing the E. coli rnt gene encoding RNase T was isolated in a screen for high copy suppressors which confer UV resistance to an E. coli strain deficient in the three known single-strand DNA exonucleases [4]
This DNase activity was specific for single-strand DNA (ssDNA); no DNase activity was detected on double-strand DNA substrates

Summary

EXPERIMENTAL PROCEDURES

Strains and Plasmids—The STL2350 strain (xonA2 recJ284::Tn10 ⌬(xseA-guaB) zff-3139::Tn10kan) was used for RNase T protein expression and purification due to its attenuated background DNase activity. The active fractions were pooled and dialyzed against Buffer A ϩ 50 mM NaCl. The dialysate, containing 0.83 mg of protein was applied to a 1-ml Econopac MonoQ cartridge (Bio-Rad) equilibrated in Buffer A ϩ 50 mM NaCl. A 24-ml linear gradient from 50 mM NaCl to 1 M NaCl in Buffer A was applied with a sharp peak of ssDNase activity eluted at 200 mM NaCl. The pooled fractions were dialyzed twice against 20 mM Tris-HCl (pH 7.5), 60% glycerol and 1 mM dithiothreitol (Buffer C) and stored at Ϫ20 °C (Fraction 6, 2.3 ml). The eluted protein was dialyzed against Buffer C and stored at Ϫ20 °C (Fraction 2, 2.0 ml)

RESULTS

A N-terminal His6-tagged RNase T protein was constructed

DISCUSSION