Functional Consequences and Exonuclease Kinetic Parameters of Point Mutations in Bacteriophage T₄ DNA Polymerase

Abstract

Three groups of T4 DNA polymerase mutants were prepared and characterized. In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I. Two divalent metal ions required for catalyzing the 3'-5' exonuclease reaction are ligated by carboxyl groups from these conserved Asp and Glu residues. The Ala and Asn replacements have a profound effect on the exonuclease activity of T4 DNA polymerase and also have a significant, but less pronounced influence on its polymerase activity which is located in a domain distal to the exonuclease region. The kcat values for the exonuclease reaction were reduced by 3-4 orders of magnitude by these replacements, but the values of Km(app) did not differ greatly from the wild-type enzyme. The second group consists of replacements of other residues, that are conserved in the exonuclease domain of eukaryotic DNA polymerases, but do not contribute to divalent metal ion coordination. Many of these alterations resulted in decreased exonuclease and/or polymerase activity. Mutants in the third group have substitutions of conserved residues in the polymerase domain which diminished polymerase and altered exonuclease activities. Our results, combined with structural data on crystals of protein N388, a truncated form of T4 DNA polymerase (Wang et al., 1996), show that: (i) the reduction in the relative specific exonuclease activities of mutants in the first group was significantly less than that of mutants in the Klenow fragment, despite the nearly identical geometric arrangement of the metal liganding groups in two proteins; (ii) altered residues, that affect exonuclease and/or polymerase activities in mutants of the second group, cluster within a small area of the exonuclease domain, suggesting that this area may be directly or indirectly involved in polymerase activity; (iii) mutations in the third group, which affect polymerase and exonuclease activities, may participate in DNA and dNTP binding. Our results point to the functional interdependence of the polymerase and exonuclease domains in T4 DNA polymerase, a property not observed with the Klenow fragment.

Identification and Expression of the TREX1 and TREX2 cDNA Sequences Encoding Mammalian 3′→5′ Exonucleases

“…Structural and mutagenesis studies of DNA pol I suggest a role for this conserved Tyr at the active site (31,32). For T4 DNA pol mutations at this Tyr affect both the polymerase and the exonuclease activities (35,39). An alternative Exo III motif, named Exo III⑀, has been proposed based on mutagenesis studies of the exonuclease domain of Bacillus subtilis DNA pol III (40).…”

Retention of replication fidelity by a DNA polymerase functioning in a distantly related environment

“…These sites were chosen for alteration because they occupy conserved Exo motifs (11,14,15). The mutant gp43s display Ͻ0.1% of the wild-type 3Ј-exonuclease activity (16) in an oligonucleotide digestion assay (17).…”

Bacteriophage T4 Genome

“…These are (i) ORFs under control of a late promoter in the clockwise direction, where almost exclusively late genes are found (5.1, 5.3, 5.4); (ii) ORFs following late promoters (some of which also may still be expressed from upstream early and/or middle promoters) in the counterclockwise direction (rI. 1 Because they are likely to be expressed immediately after infection, some of the 127 uncharacterized T4 ORFs may be involved in the transition from host to phage metabolism or in resistance to plasmid-or prophage-encoded toxic proteins. Many of these genes (shown in white in Fig.…”