Talk abstracts

Talk on Thursday 09:00-09:30am submitted by Chuck Bell

An electrostatic ratchet mechanism for processivity of λ exonuclease

Jinjin Zhang (OSBP, Department of Molecular and Cellular Biochemistry, Ohio State University), Kimberly McCabe (Ohio State Biochemistry Program, Department of Molecular and Cellular Biochemistry, Ohio State University), Charles E. Bell (Ohio State Biochemistry Program, Department of Molecular and Cellular Biochemistry, Ohio State University)

Abstract:
λ exonuclease is a Mg²⁺-dependent enzyme that binds to dsDNA ends and processively digests the 5’-ended strand to form mononucleotides and a long 3’-overhang that is a substrate for recombination. Due to its unique biochemical features, λ exonuclease is currently being exploited in powerful new biotechnology applications for genetic engineering ("recombineering") and nanopore DNA sequencing. Previous studies revealed that λ exonuclease forms a toroidal trimer with a central funnel-shaped channel for tracking along the DNA substrate, and a mechanism for processivity was proposed involving topological linkage of the trimer to the ssDNA tail generated during the reaction. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.8 A resolution. The DNA binds within the central channel as expected, but is asymmetrically tilted so that the 5’-end projects into the active site of one subunit of the trimer. The enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5’-ended strand insert into the active site cleft of one of the subunit of the trimer, while the 3’-ended strand passes through the central channel to emerge at the back of the trimer. The terminal 5’-phosphate of the DNA binds to a positively charged pocket at the end of the active site cleft, while the scissile phosphate bridges the two active site Mg²⁺ ions. Unwinding of the DNA is facilitated by several hydrophobic residues, including Leu78, which is wedged between the 2^nd and 3^rd bases of the 5’-ended strand. Our data suggests a mechanism for processivity in which Leu78 acts as a gate to prevent backward movement of the DNA substrate, while the electrostatic attraction of the 5’-phosphate generated at each step of the reaction to the positively charged pocket provides a force to drive forward movement of the enzyme along the DNA substrate. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.

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