Genetic and Crystallographic Studies of the 3′,5′-Exonucleolytic Site of DNA Polymerase I

Abstract

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

Structural aspects of protein-DNA recognition

“…16), therefore allowing editing of any single-stranded DNA sequence as would be required for error-free DNA replication . Two divalent metal ions are also seen interacting with the phosphate of the 3' terminal base and are thought to be involved in the positioning and cleavage of the phosphodiester bond (Derbyshire et al, 1988;Freemont et al, 1988). Recent site-directed mutagenesis studies of all the amino acids implicated by the crystal structure to be involved in substrate binding or catalysis are in agreement with the structural observations (Derbyshire et al, 1991).…”

“…The smaller N-terminal domain has a central core of f-sheet with ahelices on both sides and in the crystal can bind dTMP and two divalent metal ions. Various experiments (reviewed in Joyce & Steitz, 1987;Freemont et al, 1986;Derbyshire et al, 1988) have shown that the C-terminal domain contains the active site for the polymerase reaction and the N-terminal domain catalyses the 3'-5' exonuclease activity. DNA polymerase I has a strict requirement for base-paired primer-template DNA substrates and no sequence specificity, although under certain conditions single bases can be added to blunt-ended duplex (Clark et al, 1987).…”

Metal activation of synthetic and degradative activities of .vphi.29 DNA polymerase, a model enzyme for protein-primed DNA replication

“…Like the majority of DNA-dependent DNA polymerases, $29 DNA polymerase has a 3' -5' exonuclease activity (Watabe et al, 1984;Blanco & Salas, 1985b). In the case of pol I K, it has been shown that this activity resides in a different domain from the polymerase activity Derbyshire et al, 1988). On the basis of conservation of the critical amino acid residues forming the 3'-5' exonuclease active site, and site-directed mutagenesis studies, the same structural arrangement has been proposed for 429 DNA polymerase (Bernad et al, 1989;Blanco et al, 1991) and for T4 DNA polymerase (Reha-Krantz, 1989;Blanco et al, 1991).…”

“…Thus, the putative residues involved in metal binding for both polymerase and exonuclease activities appear among the best conserved ones in DNA polymerases. By mutagenesis studies (Derbyshire et al, 1988), it has been established that metal A is mainly involved in substrate binding and metal B in catalysis of the 3 ' 4 5' exonuclease reaction. The affinity of these two sites for Mn2+ has been studied quantitatively in solution (Mullen et al, 1990).…”

“…In the three DNA polymerases, synthetic and degradative activities were proposed to reside in different domains of the enzyme Bemad et al, 1989;Derbyshire et al, 1988;Reha-Krantz, 1988), and, therefore, they could be expected to show different metal ion requirements. According to that, we have studied metal activation of both synthetic and degradative activities separately.…”