Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation

Abstract

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.

FIGURE 1.

Amino acid sequences and schematic representation of RecJ-like and RecJ proteins. A, amino acid sequences of TTHA0118, Mpn140, NrnA, NrnB, SMU.1297, BF3670, ttRecJ, E. coli RecJ (ecRecJ), and B. subtilis RecJ (bsRecJ) are aligned according to Aravind and Koonin (9). The numbers of amino acids not shown in the alignments are in parentheses. Motifs with variant residues are numbered. U indicates a bulky hydrophobic residue, and O indicates a small residue. TTHA0118 (T. thermophilus HB8, YP_143384); Mpn140 (M. pneumoniae M129, NP_109828); NrnA (B. subtilis M168, NP_390803); NrnB (B. subtilis M168, NP_389702); SMU.1297 (S. mutans UA159, NP_721668); BF3670 (B. fragilis NCTC 9343, YP_213263); ttRecJ (T. thermophilus HB8, YP_144433); ecRecJ (E. coli ATCC 8739, YP_001723816); and bsRecJ (B. subtilis M168, NP_390640). B, schematic representation of TTHA0118, ttRecJ, and cd-ttRecJ. DHH and DHHA1 indicate DHH motifs I-IV and DHHA1 motif, respectively. OB indicates OB-fold.

FIGURE 2.

Exonuclease activity of TTHA0118, Mpn140, NrnA, ttRecJ, and cd-ttRecJ. An aliquot of 10 nm 5′-³²P-labeled 11-mer ssDNA (11f) was reacted with 0.1 or 1 μm TTHA0118 (tt0118), Mpn140, NrnA, ttRecJ, or cd-ttRecJ for 1 h at 37 °C. The reaction mixtures contained 50 mm HEPES, 100 mm KCl, 5 mm MnCl₂ (for TTHA0118, Mpn140, and NrnA reactions) or 5 mm MgCl₂ (for ttRecJ and cd-ttRecJ reactions) (pH 7.5). Lane 1, 5 mm MnCl₂; lane 2, 5 mm MgCl₂; lane 3, 0.1 μm TTHA0118; lane 4, 1 μm TTHA0118; lane 5, 0.1 μm Mpn140; lane 6, 1 μm Mpn140; lane 7, 0.1 μm NrnA; lane 8, 1 μm NrnA; lane 9, 0.1 μm ttRecJ; lane 10, 1 μm ttRecJ; lane 11, 0.1 μm cd-ttRecJ; lane 12, 1 μm cd-ttRecJ. The 11-mer arrow indicates the position of 5′-end labeled substrate 11-mer ssDNA, and the 1-mer arrows indicate the position of released 5′-end-labeled mononucleotides in control reactions with buffer containing Mn²⁺ or Mg²⁺ (lanes 1 and 2). Authentic 5′-end-labeled AMP was used as a marker for the position of mononucleotides.

FIGURE 3.

FT-ICR mass spectra of the products of TTHA0118, Mpn140, and NrnA exonuclease reaction. A–C, mass analysis results of the products generated by TTHA0118, Mpn140, and NrnA, respectively. The substrate (S), 5′-end phosphorylated 11-mer ssDNA, was incubated at 37 °C with each enzyme for 1 h (b, f, and j), 3 h (c, g, and k), 6 h (d, h, and l), or incubated for 6 h without enzyme (a, e, and i) as a negative control. The relative intensities of b, c and d were four times magnified. The isotopic mass distribution of 5′-end phosphorylated 11-mer ssDNA (black line) and its simulated pattern (red dotted line) are shown in the inset of panel a in A. Peak labels show x-ions by the letter x and nucleotide length by numbers written as subscripts (see supplemental Fig. S2).

FIGURE 4.

HPLC profile of the products of TTHA0118 phosphatase activity on a pAp substrate. Elution profile of the product of pAp hydrolysis by TTHA0118 (red solid line), a pAp control reaction mixture (black dotted line), a 5′-AMP (red dotted line) control reaction mixture, and a 3′-AMP control reaction mixture (black solid line). The enzyme reaction mixture was prepared as described in “Experimental Procedures.” The control reaction mixture did not contain TTHA0118.

FIGURE 5.

Phenotype of the Δttha0118 strain. Cell density after 21 h cultivation of wild-type (gray columns) and Δttha0118 (white columns) strains in liquid TT or CS medium in the absence or presence of 0.1 mm 5′-mononucleotides (5′-NMP), nucleosides (Nuc), or cysteine (Cys). Each cell density is the arithmetic mean ± standard deviation for four independent experiments.

FIGURE 6.

Structural comparison between RecJ and RecJ-like protein. The active site is surrounded by a dashed line. A, crystal structure of cd-ttRecJ (PDB ID: 1I6R). Residues thought to be important for ssDNA binding are shown in stick form (37, 38). His-394 is a residue in DHHA1 motif. B, crystal structure of BF3670 (PDB ID: 3DMA). Residues thought to be important for the substrate binding are shown in stick form. His-311 is a residue in DHHA1 motif. C, crystal structure of ttRecJ (PDB ID: 2ZXP). The OB-fold-containing domain is shown in orange. Residues thought to be important for ssDNA binding in this domain are shown in stick form (38).

FIGURE 7.

Overview of RecJ-like protein functions. RecJ-like protein has exonuclease activity for short ssDNA and ssRNA oligomers to produce mononucleotides, and/or phosphatase activity for pAp to control pAp concentration.