Elucidation of the metal-binding properties of the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase by lanthanide(III) luminescence spectroscopy.
BACKGROUND
The exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I has a double binding site for the two essential divalent metal ions in the presence of the nucleotide monophosphate dTMP.


RESULTS
The luminescence spectroscopy observed upon binding of Eu3+ to the exonuclease active site of T4 DNA polymerase was interpreted relative to the binding of Eu3+ or Tb3+ observed with KF. Both wild-type enzymes tightly bind a single Ln3+ ion but in two isomeric forms. The single mutants of KF (D424A) and T4 (D219A) also bind a single Eu3+ ion tightly, but the alignment of the coordinating ligands is altered. The KF double mutant (D355A, E357A) exhibits a markedly altered and weakened binding site (Kd = 20-26 microM). Eu3+ serves as a competitive inhibitor of Mg2+-induced polymerase and exonuclease activity, validating its use as a probe for these active sites.


CONCLUSIONS
Ln3+ luminescence spectroscopy is established as a sensitive way to determine the consequences of exonuclease binding-site mutations and to examine binding site similarities and differences among DNA polymerases from different sources. The binding sites of KF and T4 DNA polymerase are shown to be quite similar.
Frey
M W
MW
Department of Chemistry, 152 Davey Laboratory, The Pennsylvania State University, University Park, PA 16802, USA. WDH2@PSUVM.PSU.EDU
Frey
S T
ST
Horrocks
W D
WD
Kaboord
B F
BF
Benkovic
S J
SJ
D004256:DNA Polymerase I / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000201:enzymology*; D008670:Metals / Q000378:metabolism*; D008674:Metals, Rare Earth; D011485:Protein Binding; D013057:Spectrum Analysis; D014764:Viral Proteins / Q000378:metabolism*
D008670:Metals; D008674:Metals, Rare Earth; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase