EcoRI DNA methyltransferase-DNA interactions.

We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)

The corrected nucleotide sequences of the TaqI restriction and modification enzymes reveal a thirteen-codon overlap.

The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.

Cloning and sequencing of genes encoding the TthHB8I restriction and modification enzymes: comparison with the isoschizomeric TaqI enzymes.

Genes encoding the TthHB8I restriction and modification (R-M) system from Thermus thermophilus HB8 (recognition sequence T decreases CGA) were cloned in Escherichia coli. The genes have the same transcriptional orientation, with the last 13 codons of the methyltransferase (MTase) overlapping the first 13 codons of the endonuclease (ENase). Nucleotide sequence analysis of the TthHB8I ENase revealed a single chain of 263 amino acid (aa) residues that share a 77% identity with the corrected isoschizomeric TaqI ENase. Likewise, the Tth MTase (428 aa) shares a 79% identity with the corrected sequence of the TaqI MTase. This high degree of aa conservation suggests a common origin between the Taq and Tth R-M systems. However, codon usage and G+C content for the R-M genes differed markedly from that of other cloned Thermus genes. This suggests that these R-M genes were only recently introduced into the genus Thermus.

Non-additivity of sequence-specific enzyme-DNA interactions in the EcoRI DNA methyltransferase.

We describe a novel strategy to characterize protein-DNA interactions involving monomeric enzymes such as DNA methyltransferases (Mtases). This strategy is applied to our investigation of the EcoRI DNA Mtase, which binds its double stranded recognition site 5'-G-AATTC-3' and methylates the central adenosine of each strand using S-adenosyl-L-methionine as the methyl donor. We show that prior methylation of adenosine in either strand does not perturb catalysis. In contrast, substrates substituted with deoxyinosine at either guanosine position (T-BMI5 and TI5-BM) show the minor groove residing N2 amino group of both guanosines contribute to DNA recognition since specificity constants for the modified substrates are reduced 13 and 39 fold. Similar analysis of a substrate containing deoxyinosine at both positions (TI5-BMI5) clearly shows that some communication occurs between the sites. To determine the extent to which structural changes in the DNA alone contribute to this lack of additivity, we performed DNA melting analysis of the singly and doubly substituted substrates, and also found non-additivity. Although our functional and structural analyses suggest that deoxyinosine incorporation causes long range conformational effects, the similarity of KmAdoMet for all substrates suggests that no large-scale structural changes occur in the Mtase-DNA-AdoMet complex. Our results support the following conclusions: 1) The non-additivity shown in this system contrasts with the widespread demonstration of additivity involving repressors [Lehming et al., 1990; Takeda et al., 1989; Ebright et al., 1987], suggesting that sequence discrimination by enzymes may involve more complex mechanisms. Further, this non-additivity precludes quantitative assignment of individual interactions and we suggest that future analyses of this and related enzyme systems with base analogs include detailed information about the long range structural consequences of individual substitutions. 2) Although TI5-BM and T-BMI5 are shown to be radically different by thermodynamic analysis, the similar specificity constants with the Mtase suggest that the underlying structural differences (e.g., altered helical parameters of the DNA) are not critical for sequence-recognition. 3) The significance of minor groove Mtase-DNA interactions to specificity is confirmed.