Cocrystal structure of an editing complex of Klenow fragment with DNA.
High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.
Freemont
P S
PS
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Friedman
J M
JM
Beese
L S
LS
Sanderson
M R
MR
Steitz
T A
TA
D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D014961:X-Ray Diffraction
D004277:DNA, Single-Stranded; D004247:DNA; D004256:DNA Polymerase I