Mutational analysis of the RecJ exonuclease of Escherichia coli: identification of phosphoesterase motifs

Abstract

The recJ gene, identified in Escherichia coli, encodes a Mg(+2)-dependent 5'-to-3' exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of Mycoplasma and Mycobacterium tuberculosis. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including Bacillus and Helicobacter, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of E. coli RecJ. These motifs may define interactions with Mg(2+) ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of Mycoplasma, a domain of putative polyA polymerases in Synechocystis and Aquifex, PRUNE of Drosophila, and an exopolyphosphatase (PPX1) of Saccharomyces cereviseae. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.

FIG. 1

A sequence alignment with eubacterial RecJ-related sequences. Shown in parentheses are the number of amino acids not shown in the alignment. Shown in bold are residues conserved in at least 13 of the 14 eubacterial sequences. Motifs with invariant residues are numbered. Asterisks are placed above those residues mutated in this study. Sequences are listed in order of similarity to E. coli RecJ as determined by BLAST alignment (25). Eco, E. coli RecJ (PID2507106); Ech, Erwinia chrysanthemi (PID2507107); Hin, Haemophilus influenzae (PID1172895); Bsu1, B. subtilis YrvE (PID2635226); Rpr, Rickettsia prowazekii (PID3861077); Ctr, Chlamydia trachomatis (PID3328879); Ssp, Synechocystis sp. (PID2984349); Hpy1, H. pylori HP0348 (RecJ) (PID2313437); Cca, C. caviae (PID2126389); Aae, Aquifex aeolicus (PID2984349); Bbu, Borrelia burgdorferi (PID2688156); Tpa, Treponema pallidum (PID3323008); Bsu2, B. subtilis YorK (PID2634428); Hpy2, H. pylori HP0425 (PID2313533).

FIG. 2

Similarity of RecJ motifs 1 and 2 with ExoI and ExoII motifs as exhibited by members of the RAD2-FEN-1 family of 5′ exonucleases. Sequences include E. coli RecJ, T4 RNase H, Taq polymerase, T7 gene 6 protein, S. cerevisiae RAD2, human XP-G, and murine FEN-1 with respect to the published alignment of the RAD2-FEN-1 group (24).