A thermostable single-strand DNase from Methanococcus jannaschii related to the RecJ recombination and repair exonuclease from Escherichia coli

Abstract

The RecJ protein of Escherichia coli plays an important role in a number of DNA repair and recombination pathways. RecJ catalyzes processive degradation of single-stranded DNA in a 5'-to-3' direction. Sequences highly related to those encoding RecJ can be found in most of the eubacterial genomes sequenced to date. From alignment of these sequences, seven conserved motifs are apparent. At least five of these motifs are shared among a large family of proteins in eubacteria, eukaryotes, and archaea, including the PPX1 polyphosphatase of yeast and Drosophila Prune. Archaeal genomes are particularly rich in such sequences, but it has not been clear whether any of the encoded proteins play a functional role similar to that of RecJ exonuclease. We have investigated three such proteins from Methanococcus jannaschii with the strongest overall sequence similarity to E. coli RecJ. Two of the genes, MJ0977 and MJ0831, partially complement a recJ mutant phenotype in E. coli. The expression of MJ0977 in E. coli resulted in high levels of a thermostable single-stranded DNase activity with properties similar to those of RecJ exonuclease. Despite overall weak sequence similarity between the MJ0977 product and RecJ, these nucleases are likely to have similar biological functions.

FIG. 1

Comparison of RecJ motifs among archaeal proteins and PPX1 of S. cerevisiae. MJ, AF, MTH, and PHO prefixes indicate proteins of the archaea M. jannaschii, A. fulgidus, M. thermoautotrophicum, and P. horikoshii, respectively. Eco, E. coli sequence; Sce, S. cerevisiae sequence. NR, the motif is not recognizable within the sequence alignments. Each member of the defined groups has very strong sequence similarity to other members of the group, with E values in pairwise BLASTP alignments of <10⁻²⁵. (A) Conserved motifs from the E. coli RecJ sequence, with invariant residues among the eubacterial RecJ-related sequences (11) shown in bold. (B) Motifs from group B archaeal sequences with strongest similarity to RecJ. (C) AF0075 archaeal sequence with somewhat weaker similarity to RecJ and similarity to group B. (D) Archaeal RecJ-related sequences with both RecJ motifs and an N-terminal DnaJ domain. (E) AF0291, an outlying archaeal sequence with no strong homology to any of the other groups. (F) Archaeal sequences with similarity to group G. (G) Sequences with similarity to group F. (H) PPX1 of S. cerevisiae and related sequences from archaea.

FIG. 2

UV survival of E. coli STL3266 transformed with plasmids containing M. jannaschii genes, E. coli recJ, or the vector alone. Symbols: ▾, vector; ◊, E. coli recJ; ▴, MJ0831; ■, MJ0977; ●, MJ1198. Values are the means of five or more experiments.

FIG. 3

Thermostability of ssDNA nuclease activity in crude extracts of cells induced for the expression of the MJ0977 product or E. coli RecJ. Nuclease assays were performed under standard conditions (as described in Materials and Methods) with noted temperature variations. The specific ssDNA nuclease activity of extracts from cells containing the vector alone was <35 U/mg of protein.

FIG. 4

Optimization of nuclease activity on ssDNA in crude extracts of cells induced for the expression of the MJ0977 product. T7 nuclease assays were performed at 65°C under standard conditions (as described in Materials and Methods) with noted variations in Tris buffer pH (measured at 65°C) (A), NaCl concentration (B), Mg²⁺ concentration (C), or Mn²⁺ concentration in the absence of Mg²⁺ (D).

FIG. 5

DNA end preference of the expressed MJ0977 product. Values were derived by subtracting the fraction of acid-soluble radioactivity released by extracts from cells expressing MJ0977 from that released by extracts from cells containing the vector alone. Symbols: ■, 5′-end-labeled ssDNA substrate; ●, 3′-end-labeled substrate.