A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity

Abstract

Recent crystallographic studies of phi29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a phi29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of phi29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.

Fig. 1.

φ29 DNA polymerase TPR2, a specific subdomain of protein-priming DNA polymerases. (A) Location of the TPR2 insertion of φ29 DNA polymerase. A ribbon representation of the eukaryotic-type φ29 DNA polymerase structure shows its four subdomains, colored as follows: 3′-5′ exonuclease domain in red, fingers in blue, palm in pink, and thumb in green. The φ29 DNA polymerase TPR2 insertion connecting helices N (fingers) and O (palm) is indicated in cyan, and the region deleted in the ΔTPR2 mutant is represented in gray. Crystallographic data are from ref. . (B) Superposition of the homologous α-helices from fingers and palm subdomains of the eukaryotic-type DNA polymerases from bacteriophages φ29 and RB69 and E. coli DNA polymerase II, obtained by automatic fitting of the conserved Lys and Tyr residues from motif Kx₃NSxYG and Thr residue from motif Tx₂G/AR by using the program swiss-pdbviewer (www.expasy.org/spdbv). The φ29 DNA polymerase TPR2 insertion and its flanking α helices are colored in gray and blue, respectively. The corresponding helices of RB69 DNA polymerase and E. coli DNA polymerase II and the loop located between them are colored in red and yellow, respectively. Crystallographic data are from Protein Data Bank ID codes 1IG9 (RB69 DNA polymerase), 1XHX (φ29 DNA polymerase), and 1Q8I (E. coli DNA polymerase II). The amino acid side chains in ball-and-stick representation are the underlined amino acids in the Kx₃NSxYG and Tx₂G/AR motifs. (C) Amino acid sequence alignment of the region encompassing motifs Kx₃NSxYG to Tx₂G/AR of crystallized DNA-dependent DNA polymerases belonging to the eukaryotic-type (family B). The DNA polymerase nomenclature and sequences are compiled in ref. , with the exception of bacterial DNA polymerases from T. gorgonarius (GenBank accession no. P56689), P. kodakaraensis (GenBank accession no. BAA06142), Thermococcus sp.9°-N7 (23), DNA polymerase from the archaebacterial D. tok (GenBank accession no. 1QQCA), and DNA polymerase from bacteriophage RB69 (GenBank accession no. Q38087). The numbers indicate the position of the first aligned amino acid with respect to the N terminus of the respective DNA polymerase. Highly conserved residues among family B DNA polymerases are shown in red letters. Residues specifically conserved in the bacteriophage protein-primed subgroup of family B DNA polymerases are shown green. The amino acid sequence from residues Asp-398 to Glu-420, deleted in the φ29 DNA polymerase ΔTPR2 mutant, is indicated.

Fig. 2.

φ29 DNA polymerase ΔTPR2 is impaired in its DNA-binding capacity. The assay was carried out as described in Materials and Methods by using a 5′-labeled 15/33 mer as substrate, in the presence of the indicated concentrations of wild-type or mutant φ29 DNA polymerases. Samples were analyzed by polyacrylamide gel electrophoresis and autoradiography. Bands corresponding to free DNA and to the DNA polymerase/DNA complex are indicated.

Fig. 3.

The φ29 DNA polymerase ΔTPR2 mutant has both polymerization and exonuclease activities. The assay was carried out as described in Materials and Methods by using a ³²P-labeled 15/21 mer as primer/template DNA and the indicated concentrations of dNTP. Polymerase or 3′-5′exonuclease activities are detected as an increase or decrease, respectively, in the size (15 mer) of the 5′-labeled primer.

Fig. 4.

The φ29 DNA polymerase ΔTPR2 mutant shows a distributive polymerization pattern. The assay was carried out as described in Materials and Methods by using a 5′-labeled 15/33 mer as substrate, in the presence of the indicated concentrations of wild-type or mutant φ29 DNA polymerases.

Fig. 5.

Removal of the TPR2 insertion disables DNA polymerization coupled to strand displacement. The polymerization assay was carried out on a 5-nt gapped and nongapped substrate as described in Materials and Methods by using 24 or 360 nM wild-type or mutant DNA polymerases, respectively, and the indicated increasing concentration of the four dNTP. After incubation for 10 min at 25°C, the reaction was stopped, and samples were analyzed by 8 M urea/20% PAGE and autoradiography.

Fig. 6.

Modeling processivity and strand displacement in φ29 DNA polymerase. Based on the results presented here and on the crystallographic structure of φ29 DNA polymerase (19), the TPR2 insertion would contribute to a full encirclement of the DNA substrate, conferring a remarkable processivity, and also acts as a structural barrier, which would force the DNA strands of the parental DNA to diverge (melt). Because φ29 DNA polymerase translocates after each polymerization cycle, the TPR2 subdomain would act as a wedge to couple polymerization to strand displacement. φ29 DNA polymerase subdomains are colored as indicated in Fig. 1 A. Modeled DNA is colored as follows: growing primer strand in gray, template strand in yellow, and displaced strand in green.