The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

Abstract

The a-like DNA polymerases from bacteriophage 429 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser domain (10, 11, 13, 14). In contrast, the N-terminal portion contains three regions of lower amino acid similarity that are also shared by DNA polymerases of other groups; it has been proposed (22) that these regions, by structural and functional homology with the Escherichia coli DNA polymerase I (pol I), form a conserved 3' --5' exonuclease active site.The high conservation of the YGDTDS motif in a-like DNA polymerases from distantly related organisms suggests that the YGDTDS motif has a functional role; however, the lack of direct biochemical data has until now made the catalytic significance of the YGDTDS motif rather speculative. A similar amino acid sequence motif, Tyr-(Gly/ Met)-Asp-Asp, is present in other DNA or RNA polymerases that are either DNA or RNA dependent (21). This homology and the finding that amino acid substitutions within the Tyr-Gly-Asp-Asp motif of QB replicase reduced its activity in vivo (23) led to the proposal of a catalytic role ofthe YGDTDS motif in polymerization (21). The fact that the YGDTDS sequence is not restricted to RNA-or DNA-primed DNA polymerases but is also present in DNA polymerases for terminal protein-containing DNA genomes is of additional interest because it was not known whether the specific protein-priming activity of these DNA polymerases had its own catalytic site or shared general domains of the polymerization active site. Bacteriophage 429 DNA polymerase, product of viral gene 2 (Mr, 66,520), initiates replication (24-26) by catalyzing the formation of a phosphoester bond between the 8-OH group of Ser-232 in the terminal protein (p3) and 5'-dAMP, the 5' terminal nucleotide at both DNA ends (27). The 429 DNA polymerase also has 5' -* 3' polymerization and 3'-* 5' exonuclease activities characteristic of other DNA replicases.Here we report results of site-directed mutagenesis in the most conserved C-terminal motif (YGDTDS) and its effect on the various enzymatic activities of the 429 DNA polymerase (for a preliminary report of some of these findings, see ref.28). Based on these results and the amino acid sequence similarity with other metal binding enzymes, we propose a functional role for this motif in the 429 DNA polymerase and other a-like polymerases. MATERIALS AND METHODSNucleotides, Proteins, and Templates. Unlabeled nucleotides were purchased from Pharmacia P-L Biochemicals.[a-32P]dNTPs (410 Ci/mmol; 1 Ci = 37 GBq) were obtained from Amersham. 429 terminal protein (p3) and protein p6 were purified as described (29,30 (22).Activity Assays. The initiation reaction (i.e., protein p3-dAMP complex formation), DNA polymerase "minimal" Abbreviations: YGDTDS, Tyr-Gly-Asp-Thr-Asp-S...

φ29 Family of Phages

“…For several DNA polymerases, including the 29 DNA polymerase, it has been demonstrated that three Asp residues form a metal binding triad required for catalysis at the polymerization active site (reviewed in reference 32). In the 29 DNA polymerase, the three Asp residues implicated are Asp 249 , belonging to motif A, and Asp 456 and Asp 458 , both belonging to motif C (21,35,185). These three Asp residues are conserved in the DNA polymerases of B103 and GA-1 and in all other known members of the B-type DNA polymerases.…”

“…5, together with the amino acid sequence corresponding to each motif present in the DNA polymerase of 29, B103, and GA-1. Site-directed mutagenesis at motifs A, B, and C of 29 DNA polymerase (21,(34)(35)(36) showed that these three regions form an evolutionarily conserved polymerization-active site. Figure 5 shows that of these three motifs, only motif C is fully conserved in the DNA polymerases of B103 and GA-1.…”

Polymerase structures and function: variations on a theme?

“…The available evidence suggests that the sequence alignment of Delarue et al (16) provides a reasonable starting point for the identification of active-site residues. Thus, site-directed mutagenesis studies have demonstrated the importance of the carboxylates of motifs A and C in several members of the DNA polymerase ␣-family (6,7,12,22,24) and one RNA-dependent RNA polymerase (39). The Pol ␤ structure hints at the variety of different palm subdomain structures that may be found in polymerases from nonhomologous families, though until more nonhomologous polymerase structures have been solved, we cannot predict how many different ways there may be of constructing a palm subdomain.…”

The translesion polymerase ζ has roles dependent and independent of the nuclease MUS81 and the helicase RECQ4A in DNA damage repair in Arabidopsis

“…Studies with the Bacillus subtilis phage F29 DNA polymerase and human polymerase alpha have indicated that mutations of the first Asp result in a partially functional polymerase with a deficiency in processivity. Motif C, however, is involved in both the initiation and elongation reactions (Bernad et al, 1990;Copeland and Wang, 1993). As expected, we were able to show that expression of the wild-type REV3 construct complemented the hypersensitivity of rev3-2 to MMC-, MMS-, cisplatin-, and UV-B-induced DNA damage in Arabidopsis.…”

“…We also mutated an Asp to Ala in motif C (D1549A). Bacterial polymerases harbor an identical YGDTDS motif found in REV3, and biochemical studies have shown that the respective mutation in the a-like DNA polymerase of the phage Phi29 results in a partially active enzyme with reduced elongation activity (Bernad et al, 1990). Thus, we expected that this mutation should not result in a total knockout of REV3 function but mainly a defect in the translocation step, such that this enzyme should still be able to incorporate single nucleotides.…”