Molecular cloning and functional analysis of the MutY homolog of Deinococcus radiodurans

Abstract

The mutY homolog gene (mutY(Dr)) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY(Ec)) protein. Expressed MutY(Dr) is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY(Dr) with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY(Ec) protein, purified recombinant MutY(Dr) expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY(Dr) exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY(Dr) at the position corresponding to the position of MutY(Ec) Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY(Dr) are similar to those of MutY(Ec). Although MutY(Dr) has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY(Ec) does, the binding affinities for both mismatches are slightly lower for MutY(Dr) than for MutY(Ec). Thus, MutY(Dr) can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.

FIG. 1

Comparison of amino acid sequences of MutY_Dr and MutY_Ec. Identical and conserved residues are indicated by black and gray boxes, respectively. The sequences used were the sequences of E. coli MutY (EcMutY) (accession no. P17802) and D. radiodurans MutY (DrMutY) (accession no. NC 001263). The conserved Asp residue is indicated by a dot. The position of Ser120 of MutY_Ec and Tyr134 of MutY_Dr at the conserved Lys downstream of the HhH motif of the HhH family is indicated by an asterisk. A triangle indicates the position of Lys142 of MutY_Ec and Arg156 of MutY_Dr.

FIG. 2

SDS-polyacrylamide gel analysis of MutY_Dr. The proteins were separated on a 12% polyacrylamide gel in the presence of SDS and stained with Coomassie blue. Protein markers (Gibco BRL prestained protein marker) were run in lane 1. Lanes 2 to 9 contained fractions I (11 μg; crude cell extract), II (11 μg; fraction after streptomycin sulfate treatment), III (7.3 μg; fraction after ammonium sulfate treatment), IV (3 μg; fraction after phosphocellulose column elution), V (1.8 μg; fraction after hydroxylapatite column elution), VI (1.5 μg; fraction after MonoQ column elution), VIIc (1.5 μg; fraction after MonoS column elution), and VIIc (7.5 μg; fraction after MonoS column elution), respectively. Excess protein was loaded in lane 9 to show the degree of homogeneity.

FIG. 3

Effects of EDTA on MutY_Dr binding and glycosylase activities with A/G- and A/GO-containing DNA. (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY_Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY_Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).

FIG. 4

Glycosylase activities of MutY_Dr (DrMutY) and MutY_Ec (EcMutY) with different mismatches. Oligonucleotide substrates (3′-end-labeled 20-mers; 1.8 fmol) containing the mismatches indicated above the lanes were incubated with 3.6 nM MutY_Dr (lanes 1 to 8) or 3.6 nM MutY_Ec (lanes 9 to 16) for 30 min at 37°C. Abbreviations: O, GO; 2, 2-aminopurine. The arrows indicate the positions of intact DNA substrate (arrow I) and the cleaved DNA fragment (arrow N). The minor band above the cleaved product was derived from an impurity of DNA substrates that appeared above the intact DNA.

FIG. 5

MutY_Dr exhibits limited AP lyase activity. (A) Glycosylase reactions performed under different reaction conditions. Lane 1, sample supplemented with 5 μl of formamide dye and directly loaded onto a gel that was electrophoresed at 1,000 V without drying and heating; lane 2, sample supplemented with 5 μl of formamide dye and heated at 90°C for 2 min before it was loaded onto a gel without drying; lane 3, sample treated with 1 M piperidine at 90°C for 30 min after the reaction, dried, resuspended in 3 μl of formamide dye, and heated at 90°C for 2 min; lane 4, DNA alone. The samples were from concurrent experiments, but the products were separated on nonadjacent lanes in one sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I), products with a 3′-α,β-unsaturated aldehyde formed via β-elimination (arrow B), and products with a 3′ phosphate formed via β/δ-elimination by piperidine (arrow P). (B) Formation of covalent complexes of MutY_Dr and A/GO-containing DNA in the presence of various concentrations of NaBH₄. Lane 1, reaction mixture containing 72 fmol of E. coli MutY in MutY_Ec buffer (20 mM Tris-HCl [pH 7.6], 1 mM dithiothreitol, 1 mM EDTA, 2.9% glycerol) in the presence of 100 mM NaBH₄; lanes 2 to 11, reaction mixtures containing 14.4 fmol of MutY_Dr with different concentrations of NaBH₄ (100, 80, 60, 50, 40, 30, 20, 15, 10, and 5 mM, respectively). The products after heating at 90°C for 2 min were fractionated in an SDS–12% polyacrylamide gel. The positions of free oligonucleotide (arrow F) and a covalent complex (arrow C) are indicated by arrows.

FIG. 6

Time course studies of MutY_Dr glycosylase activities with A/G- and A/GO-containing DNA. A/G-containing (▪) and A/GO-containing (▴) 20-mer oligonucleotides (1.8 fmol) were incubated at 37°C with 72 fmol of MutY_Dr for various times. After reaction, the products were treated with piperidine, dried, resuspended in formamide dye, heated at 90°C for 2 min, and analyzed on a 14% polyacrylamide denaturing sequencing gel. Data were obtained from PhosphorImager quantitative analyses of gel images from three experiments. The percentages of DNA cleaved were plotted as a function of time.