Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage.

DNA bending is a common attribute of protein-nucleic acid complexes despite the expenditure of free energy required to achieve the bent state (1). Examples of DNA bending in biologically important contexts include the relatively smooth curvature in nucleosomes that facilitates packaging, and the often sharp and localized bending by transcription factors to juxtapose two segments of DNA that are far apart in sequence (2-6). DNA bending is also known to be important to DNA replication, repair, recombination and methylation (7-17). Many DNA bending proteins are also site-specific enzymes, so that the bending event is coupled to selective enhancement of the reaction rate at cognate sites. For example, restriction enzymes enhance the rate of DNA strand scission at specific sites by an estimated 1015-fold (18). The specificity of these enzymes is exquisite - a single incorrect base pair in a 4 to 6 base-pair target site reduces kcat/KM by 106-fold or more (19-21). Furthermore, these enzymes must also bind to nonspecific sites, prior to the one- and three-dimensional search for the cognate sequence within a vast molar excess of nonspecific DNA (22-24). In this context, the extent of DNA bending at specific but not nonspecific sites likely provides a basis for sequence discrimination, because enzyme complexes at nonspecific sites provide insufficient interaction energy to drive the unfavorable bending transition. Thus, free energy liberated in the highly complementary specific complex can be used to drive the conformational change which assembles the active site, thereby lowering the energy of the transition state for cleavage. In contrast, the incomplete conformational change associated with binding to nonspecific sites may result in the improper juxtaposition of reactive moieties for the subsequent catalytic steps (25, 26). EcoRV restriction endonuclease cleaves the phosphodiester backbone of DNA at the center TA step of its recognition sequence 5’-GATATC (27, 28). For some years the enzyme has served as an important model system to explore the importance of DNA bending in specificity and catalysis. EcoRV sharply bends its specific DNA site by approximately 50° directly at the center TA step (29), helping to facilitate proper juxtaposition among the scissile phosphate, the catalytic side chains Asp90, Asp74 and Lys92, and divalent metal ions. DNA bending also produces a severe narrowing of the major groove at the center TA step, preventing the enzyme functional groups from penetrating to make discriminating hydrogen bonds with the bases. Instead, the center step is recognized primarily by indirect readout (30). The operation of indirect readout as a mechanism that underlies specificity provides another distinctive feature driving the sustained interest in EcoRV as a model system. Comparisons among a number of EcoRV structures bound to specific DNA reveal that, as the bend angle of the DNA increases, more surface area is buried at the protein-nucleic acid interface (31). In this analysis, it was suggested that direct contacts between the DNA binding domains of EcoRV and DNA provide a force to facilitate DNA bending, via direct contacts with symmetrically disposed ribose sugars in the minor groove. However, in EcoRV electrostatic interactions on the concave surface of the DNA may also induce DNA bending into the major groove via asymmetric phosphate neutralization (32-36). By this mechanism, appropriately positioned positive charge along one face of the duplex promotes bending of the DNA in that direction, by allowing compensation of the unfavorable closer juxtaposition of phosphates on the inside of the bend. In the proposed two-stage model for EcoRV bending, the DNA is first bent into the major groove by the operation of asymmetric phosphate neutralization, which could function even at nonspecific sites during the course of target localization. After the specific site is reached, formation of the complementary enzyme-DNA interface, including the discriminatory contacts with base-specific moieties, would drive formation of the complete sharp localized bend at the center TA step (31). A significant proportion of the positively charged surface of EcoRV is found in the 29 amino acid C-terminal subdomains, which possess a total net +4 charge (Figure 1). Two basic amino acids from the subdomain, Arg221 and Arg226, make direct contacts with phosphates within and flanking the GATATC target site. The subdomains are located on the concave DNA major groove side, and are thus in an appropriate position to contribute to asymmetric phosphate neutralization both by direct phosphate contacts and by the influence of diffuse charge. To understand the role of the C-terminal subdomain in DNA binding, bending, and catalysis, we carried out a thorough analysis of the structural and kinetic properties of a mutant enzyme in which the subdomains were removed in their entirety (EcoRVΔC). Remarkably, the cleavage rate of the truncated enzyme is diminished by only 100-fold. However, both the rates of DNA association and dissociation, and the total extent of equilibrium DNA bending, were strongly affected. The X-ray crystal structure of EcoRVΔC bound to DNA and calcium ions showed no global perturbations in the quaternary or tertiary fold as compared with wild-type EcoRV, but suggested that divalent metal binding at the active site may be significantly destabilized. The experiments suggest a significant role for asymmetric phosphate neutralization in promoting the induced-fit transition of the EcoRV-DNA complex, at the level of both protein-DNA association and DNA bending. Figure 1 Structure of EcoRV endonuclease