Cytosine methylation by the SuaI restriction-modification system: implications for genetic fidelity in a hyperthermophilic archaeon

Abstract

5-methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles. To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated. Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S. acidocaldarius DNA. In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs. The results show that the SuaI system uses N(4) methylation to block cleavage of its recognition site, thereby avoiding the creation of G. T mismatches by spontaneous deamination at extremely high temperature.

FIG. 1.

Discrimination of GGCC methylation by isoschizomers of SuaI. Fluorescent oligonucleotide duplexes were incubated with excess endonuclease and resolved on polyacrylamide gels as described in the text. Lanes: un, unmethylated site; 5i, methyl group on carbon 5 of the inner C residue; 5o, methyl on carbon 5 of the outer C; 4i, methyl group on nitrogen 4 of the inner C; 4o, methyl group on nitrogen 4 of the outer C. (A) Fifty units of HaeIII; (B) 50 U of EsaBC4I.

FIG. 2.

Specific resistance of S. acidocaldarius DNA to GGCC-targeted endonucleases. S. acidocaldarius DNA was treated with 10 U of the indicated endonucleases and electrophoresed in an agarose gel. Abbreviations (recognition sequence): M, molecular size markers (numbers at left are numbers of base pairs [in thousands]); Hin (GCGC), HinPII; Hae (GGCC), HaeIII; con, control (no enzyme); BC4 (GGCC), EsaBC4I; Bst (CGCG), BstUI. Incubations with BstUI were at 60°C; all other incubations were as described in the text.

FIG. 3.

Methylation sensitivity of SuaI. Fluorescent oligonucleotides were digested with the indicated amounts of partially purified SuaI or commercially available HaeIII. (A) SuaI; (B) HaeIII. Lane abbreviations and other conditions were as described for Fig. 1. (C) Three micrograms of EsaBC4I-modified DNA of E. coli was incubated for 2 h at 70°C with 15 μl of S. acidocaldarius extract (equivalent to about 100 U of SuaI); other the conditions were as described in the legend to Fig. 2. Lane designations indicate NEB buffers 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂), 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂), 4 (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate), or SalI (150 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂) (b2, b3, b4, and bS, respectively). All buffers were at pH 7.9 and included 1 mM dithiothreitol. The controls were incubated without S. acidocaldarius extract (con) or preincubated for 1 h at 37°C with 30 U of HindIII (+Hin). Lane M, molecular size markers.

FIG. 4.

Relative efficiency on GG5mCC. Unmodified and M.HaeIII-modified pUC19 DNAs were treated with the indicated amounts (in microliters) of SuaI (cell extract). Methylated DNA is indicated by “m”; arrowheads identify equivalent degrees of partial endonuclease digestion.