Title: Identification of R/KRRY motif in Klenow Fragment of <i>E. coli</i> DNA polymerase I: Its role in coordinating polymerase and exonuclease activity
Abstract:The Klenow Fragment (KF) of E. coli DNA polymerase I has two activities on a single polypeptide chain-the polymerase and the 3′-5′exonuclease. These activities collectively function to enhance an erro...The Klenow Fragment (KF) of E. coli DNA polymerase I has two activities on a single polypeptide chain-the polymerase and the 3′-5′exonuclease. These activities collectively function to enhance an error-free synthesis of DNA. The active sites for the two activities however are separated by a distance of ~30Å. Thus, a movement of primer strand containing terminal mismatch would require 30Å movement. This may further involve repositioning single stranded template overhang to facilitate the movement. In order to understand the structural elements that are involved in coordination between polymerase and the exonuclease activity we examined crystal structures of KF and related enzymes with the primer bound in either exonuclease or polymerase mode. Superposition of the two structures revealed that template overhang requires to shift its position by ~ 20Å and that it is in the close vicinity of positively charged residues. These residues most likely bind to template overhang and permit primer strand motion to the exonuclease site. Because of the conservation of this cluster in a number of pol I related enzymes we have named it as R/KRRY motif. To assess this postulate, we generated mutants of the residues R822A, Y824A and a double mutant R822AY824A. Preliminary examination of the properties of these mutant enzymes has shown that the polymerase activity is relatively unchanged but their exonuclease activity on ssDNA and DNA with 1 and 3 mismatches is significantly reduced (4–14 fold reduction). Biochemical characterization of the mutant enzymes is in progress to determine the defective step. This study would establish the role of the R/KRRY motif residues in stabilizing the template strand when the primer binds in the exonuclease mode. Support: NIGMS36307Read More