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

AUTOR(ES)
RESUMO

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.

ACESSO AO ARTIGO

Documentos Relacionados