The energetic basis of specificity in the Eco RI endonuclease-DNA interaction

Citation metadata

Date: Nov. 9, 1990
From: Science(Vol. 250, Issue 4982)
Publisher: American Association for the Advancement of Science
Document Type: Article
Length: 8,909 words

Document controls

Main content

Article Preview :

High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent and conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavorable steric apposition between a group on the protein and an incorrectly placed functional group on base disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contacts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence.

Proteins that interact with particular target sequences in DNA may show sequence selective ranging from stringent to fairly permissive, depending on the requirements imposed by their functions. Extreme selectively is exemplified by restriction endonucleases, which must efficiently cleave small(4 to 6 base pairs) recognition sites on foreign DNA, but must avoid potentially lethal cleavage of the cellular genome at sites that differ by as little as one base pair. By contrast, gene-regulatory proteins bind at larger sites (12 to 30 bp) and some bind a series of related sites in a graduated fashion[1,2].

Sequence specificity is determine in part by protein contrast to the DNA bases (direct readout). Structural studies of the Eco RI endonuclease-DNA complex show that both strands of the GAATTC site are recognized by hydrogen bonds with each purine base[3,4] and contracts to the pyrimidines[4]. However, the tightly complementary surfaces in DNA-protein complexes[3-6] also included extensive contracts to the DNA backbone.

It has been suggested that the sequence-dependence of DNA conformation[7] might provide an indirect readout[6,7] by affecting the attainment of optimal complementarity both for protein-base hydrogen bonding[8] and for the precisely positioned[3,5,6] interactions between protein and DNA phosphates. However, there has been no evidence to indicate which phosphate interactions are indispensable to recognition, which make only nonspecific contributions to binding free energy, and which (if any) are altered when a protein interacts with a closely related but incorrect site. For Eco RI endonuclease, it has been suggested[9] that indirect readout may also contribute to specificity because the energy required to attain the "kinked' DNA conformation in the complex[3] is least unfavorable for the GAATTC site.

To determine the roles of direct protein-DNA contrast and of sequence-dependent DNA conformation, we analyzed the energetics of stringent discrimination, using a rigorous measure of the Eco RI-DNA interaction in transition state complexes. We manipulated the DNA side of the interface to explore those factors most likely to be generally applicable to other DNA binding proteins, and to identify...

Source Citation

Source Citation   

Gale Document Number: GALE|A9173706