Exonuclease X of Escherichia coli: A NOVEL 3′-5′ DNase AND DnaQ SUPERFAMILY MEMBER INVOLVED IN DNA REPAIR

Mohan Viswanathan,Susan T Lovett

doi:10.1074/jbc.274.42.30094

Abstract

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.

Highlights

DNA exonucleases are critical for DNA replication, repair, and recombination
We recently reported that RNase T of E. coli, previously described as a 3Ј-5Ј ribonuclease (19 –21), possessed a potent 3Ј to 5Ј distributive single-strand DNA-specific exonuclease activity [22]
Overexpression and Purification of a DNase Activity Associated with Exonuclease X—The E. coli open reading frame designated yobC, hereafter known as exoX, was amplified by PCR and cloned directionally into pBSSKϪ, placing its expression under T7 promoter control

Summary

A NOVEL 3Ј-5Ј DNase AND DnaQ SUPERFAMILY MEMBER INVOLVED IN DNA REPAIR*

Exonuclease X is a member of a superfamily of proteins that have homology to the 3؅-5؅ exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. Other family members include the bacterial proteins RNase T, RNase D, exonuclease I (ExoI), oligoribonuclease [3], the Saccharomyces cerevisiae PAN2 protein, and the human Werner syndrome protein (WRN) [1, 2, 4] These proteins share a conserved tripartite set of “Exo” motifs containing negatively charged aspartate and glutamate residues [5]. These hallmark residues can be visualized in the crystal structure of the Klenow (proofreading) subunit of E. coli DNA polymerase I to

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION