Chemistry Colloquia (hosted by Jack Freed)

The talk will focus on the use of ESR to examine the relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme. The EcoRI endonuclease, a 62 kDa homodimer, recognizes the DNA site 5’-GAATTC-3’ and cleaves both DNA strands in the presence of the catalytic cofactor Mg2+. EcoRI achieves its extraordinarily high specificity in two distinct stages: First, it binds to GAATTC as much as 90,000 fold better than at sites that differ by even one base pair, even in the absence of Mg2+. Second, in the presence of Mg2+, catalytic rates for the chemical steps of DNA cleavage are as much as 106–fold higher for the correct GAATTC site than for incorrect sites. The talk will describe a novel triangulation procedure using long range distance measurements by ESR to establish the identities of key amino acids that coordinate to Cu2+ in endonuclease EcoRI. We find that Cu2+ binding at this hitherto unknown coordination site stimulates endonuclease-DNA binding, but profoundly inhibits the Mg2+ catalyzed DNA cleavage activity of EcoRI. The results are remarkable given that Cu2+ binds 13 Å away from the Mg2+ positions in the catalytic centers. Molecular dynamics simulations suggest a novel model for inhibition of catalysis, whereby the Cu2+ ions alter critical protein–DNA interactions and water molecule positions in the catalytic sites. Taken together, the results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of enzymes.