Insights into Strand Displacement and Processivity from the Crystal Structure of the Protein-Primed DNA Polymerase of Bacteriophage φ29

Molecular Cell, Vol. 16, 609–618, November 19, 2004, Copyright 2004 by Cell Press

Insights into Strand Displacement and Processivity from the Crystal Structure of the Protein-Primed DNA Polymerase of Bacteriophage φ29 Satwik Kamtekar,1,5 Andrea J. Berman,1,5 Jimin Wang,1 Jose´ M. La´zaro,4 Miguel de Vega,4 Luis Blanco,4 Margarita Salas,4 and Thomas A. Steitz1,2,3,* 1 Department of Molecular Biophysics and Biochemistry 2 Department of Chemistry 3 Howard Hughes Medical Institute Yale University New Haven, Connecticut 06520 4 Centro de Biologı´a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Auto´noma Canto Blanco 28049 Madrid Spain

Summary The DNA polymerase from phage φ29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of φ29 DNA polymerase determined at 2.2 A˚ resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of φ29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA. Introduction All DNA polymerases require primers since, unlike RNA polymerases, they cannot initiate synthesis de novo. Because oligonucleotide synthesis has an absolute 5⬘ to 3⬘ polarity, the synthesis of the 5⬘ terminal bases of linear genomes and plasmids presents a problem. One solution to this problem is to attach the initial 5⬘ nucleotide to a hydroxyl of a protein side chain. This use of a protein as a primer has been widely employed by a number of bacteriophages, linear plasmids, and viruses, including adenovirus, poliovirus, and hepatitis B, as well as by bacteria of the genus Streptomyces (Salas, 1991; Salas et al., 1996; Chen, 1996). φ29, a 19.2 kb double-stranded DNA (dsDNA) bacteriophage of B. subtilis, is the system in which protein*Correspondence: [email protected] 5 These authors contributed equally to this work.

primed DNA replication has been studied in most detail (Salas, 1991; Salas et al., 1996). The two 5⬘ termini of the φ29 linear genome are covalently linked to a protein named terminal protein (TP) (Salas et al., 1978). The DNA polymerase and TP of φ29 form a tightly associated heterodimer in the absence of DNA (Blanco et al., 1987). Replication is initiated when polymerase forms a covalent bond between the initial dAMP and Ser232 of TP (Blanco and Salas, 1984; Hermoso et al., 1985). The template for the addition of this first nucleotide is the second base from the 3⬘ end of either strand of the double-stranded genome and is contained within a 6 base pair inverted repeat (Escarmı´s and Salas, 1981) that constitutes an origin of replication. This first nucleotide must therefore “slide back” prior to further elongation (Me´ndez et al., 1992). After the addition of 10 nucleotides, polymerase dissociates from TP and processively copies the rest of the phage genome (Blanco and Salas, 1985; Blanco et al., 1989; Me´ndez et al., 1997). In bacteriophage φ29, initiation from the twin origins of replication appears not to be synchronized, and there is no equivalent to lagging strand synthesis. As it proceeds from a given origin of replication, polymerase thus generates a long stretch of displaced single-stranded DNA (ssDNA). This stretch is protected from degradation by the phage single-strand DNA binding protein until it is eventually used as template by the polymerase proceeding from the opposite origin of replication, reforming duplex DNA (Inciarte et al., 1980; Gutie´rrez et al., 1991). The net consequence of a semiconservative cycle of replication in φ29 is, therefore, the generation of an additional genome, covalently linked at each of its 5⬘ termini to a molecule of TP. On the basis of sequence similarity, DNA-dependant DNA polymerases have been divided into six families (A, B, C, D, X, and Y [Ohmori et al., 2001; File´e et al., 2002]). Eukaryotic pol ␣, E. coli pol II, archaeal replicative polymerases, as well as the polymerases encoded by bacteriophages such as T4 and φ29, are members of the B family. A structure-based sequence alignment of φ29 DNA polymerase with bacteriophage RB69 DNA polymerase, E. coli pol II, and D. tok polymerase is included in the Supplemental Data accompanying this article (at http://www.molecule.org/cgi/content/full/16/4/ 609/DC1/). Like many other members of this family, φ29 DNA polymerase possesses both 3⬘ to 5⬘ exonucleolytic and 5⬘ to 3⬘ synthetic activities. However, in addition to binding a standard nucleic acid primer:template pair during elongation, the polymerase active site must also be able to accommodate TP:template DNA during protein-primed initiation. Extension of the primer strand during initiation leads to dissociation of TP from the polymerase. The dual ability of φ29 DNA polymerase to use either a nucleic acid or a protein as a primer has been the subject of mutational studies (reviewed by Blanco and Salas, 1996). The robust strand displacement activity and high processivity of φ29 DNA polymerase precludes the need for replication accessory factors such as helicases or clamps. When an oligonucleotide primer and a template

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uct duplex DNA and thereby also act as a clamp to enhance processivity. Results and Discussion

Figure 1. Ribbon Representation of the Domain Organization of φ29 DNA Polymerase The exonuclease domain is shown in red, the palm in pink, TPR1 in gold, the fingers in blue, TPR2 in cyan, and the thumb in green. D249 and D458, which provide the catalytic carboxylates of the polymerase active site, are shown using space-filling spheres.

consisting of 7.4 kb of circular M13 DNA are incubated with the enzyme, products over 70 kb long are produced by rolling circle replication (Blanco et al., 1989). As a consequence of its intrinsic proofreading activity, strand displacement, and processivity, φ29 DNA polymerase has been developed commercially as a tool for isothermal rolling circle DNA amplification (Dean et al., 2001a) and whole-genome amplification (Dean et al., 2001b). The structure of φ29 DNA polymerase provides insights into the functions of protein-primed DNA polymerases (Figure 1). The ability of these DNA polymerases to bind to terminal proteins is associated with two insertions into the sequence of the polymerase domain termed terminal protein regions 1 and 2 (TPR1 and TPR2) (Blasco et al., 1990; Dufour et al., 2000). TPR1 forms a novel domain, and unexpectedly, TPR2 has structural similarities to the specificity loop of T7 RNA polymerase (Cheetham et al., 1999), a member of the A, or pol I, family of polymerases. Homology modeling of primer:template DNA substrate from the structure of a ternary complex of RB69 DNA polymerase (Franklin et al., 2001) indicates that the downstream template DNA bound by φ29 DNA polymerase passes through a tunnel before entering the polymerase active site. Since this tunnel is only large enough to allow the passage of a single strand of DNA, it suggests a structural basis for both the intrinsic strand displacement and processivity of the polymerase. Finally, the polymerase has an unusual thumb, which appears to be positioned so that it may function, in concert with the polymerase palm subdomain and with inserted sequences TPR1 and TPR2, to encircle upstream prod-

Structure Determination and Overview We initially solved the structure of φ29 DNA polymerase in a monoclinic crystal form using a combination of MAD and MIR techniques. Most of these crystals exhibited an unusual lattice translocation defect, similar to that first observed in studies of imidazole methaemoglobin (Bragg and Howells, 1954). We therefore developed an algorithm to correct for this defect computationally by modifying the intensities of the observed reflections (J.W., S.K., A.J.B, and T.A.S., unpublished data). One crystal that had been incubated in a solution containing (dT)5 prior to cryoprotection diffracted to 2.2 A˚ and exhibited minimal defects. The structure was refined against data from this crystal to yield an Rcryst of 24.3% and Rfree value of 27.7% (Table 1). The asymmetric unit in this crystal form contains two crystallographically independent copies of the polymerase; all residues except the first four were built in both copies. The electron density corresponding to the bound (dT)5 oligonucleotide was of poor quality, and only three bases were positioned in the exonuclease active site. Subsequently, using molecular replacement we determined the structure of an orthorhombic crystal form, which contained four crystallographically independent copies of the polymerase. The data from this crystal form extended to 2.35 A˚ resolution, and the structure was refined to an Rcryst of 20.6% and Rfree of 25.7% (Table 1). Residues 5–575 have been built in all four copies. Incubation of the orthorhombic crystal form with (dT)5 yielded data to 2.7 A˚ and clear density corresponding to bound oligonucleotide (Figure 2). Overall, the structures in both crystal forms are extremely similar. The root-mean-squared deviations (rmsd) calculated between any two of the six crystallographically independent copies in the monoclinic and orthorhombic crystal forms using all C␣ atoms including loop regions range from 0.8 to 1.7 A˚. φ29 DNA polymerase consists of an N-terminal exonuclease domain (residues 5–189) and a C-terminal polymerase domain (residues 190–575) as shown in Figure 1. It lacks the N-terminal regulatory domain observed in the other B family polymerases whose structures have been determined (Wang et al., 1997; Zhao et al., 1999; Hopfner et al., 1999; Rodriguez et al., 2000; Hashimoto et al., 2001). Nevertheless, the structures and relative orientations of the exonuclease and polymerase domains fit well within the overall architecture of B family polymerases first described for the structure of RB69 DNA polymerase (Wang et al., 1997). Exonuclease Domain The exonuclease domains of DNA polymerases are structurally conserved across the A, B, and C families (Bernad et al., 1989) and share a common mechanism catalyzed by two metal ions that are bound to the enzyme via four carboxylate groups (Freemont et al., 1988; Beese and Steitz, 1991). The structure of the φ29 DNA polymerase exonuclease domain supports sequence comparisons and mutational data that have shown the

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Table 1. Crystallographic Statistics Data and Phasing

Data Set

Wavelength Resolution (A˚) (A˚) Rmergea (%) I/␴b

Monoclinic

(P21: a ⫽ 60.6 A˚, b ⫽ 170.6 A˚, c ⫽ 68.8 A˚, ␤ ⫽ 107⬚)

(dT)5

0.9760

50.0–2.2 9.7 (2.3–2.2) Native 1 0.9474 30.0–2.8 12.4 (2.9–2.8) Native 2 1.0080 40.0–2.3 11.4 (2.4–2.3) Hg Peak 1.0065 50.0–2.3 7.6 (2.4–2.3) Inflection 1.0087 50.0–2.5 7.3 (2.6–2.5) Remote 0.9919 50.0–2.7 7.7 (2.8–2.7) Pb Peak 0.9497 50.0–3.0 14.8 (3.1–3.0) Remote 0.9509 50.0–3.0 18.6 (3.1–3.0) Se Peak 0.9789 50.0–3.2 10.5 (3.3–3.2) Inflection 0.9793 50.0–3.7 10.7 (3.8–3.7) Figure of merit (SIGMAA combined) 0.49 (50–3.5 A˚) Figure of merit (RESOLVE) 0.53 (30–2.5 A˚)

Completeness (%)

12.4 (2.0) 99.8 (98.5)

66,838

—

—

—

9.1 (2.3)

93.0 (93.4)

29,581

—

—

—

7.0 (1.3)

96.1 (88.2)

108,178

—

—

—

9.4 (1.7)

98.1 (99.2)

116,023

0.58

0.74

20–3.5

10.0 (1.6) 98.1 (98.8)

90,034

—

—

—

8.5 (1.7)

92.5 (95.0)

67,872

0.48

0.66

20–3.5

4.6 (1.4)

89.3 (82.5)

45,986

0.92

1.16

30–4.0

3.8 (0.8)

87.6 (78.5)

47,835

0.75

0.94

30–4.0

5.1 (2.1)

77.7 (54.2)

33,403

0.51

0.48

30–4.0

5.0 (2.8)

74.3 (70.1)

24,889

0.36

0.34

20 4.0

17.0 (1.7) 99.9 (100.0)

119,984

—

—

—

14.3 (1.0) 98.0 (88.7)

78,322

—

—

—

Rmsd Bond Angle (⬚)

PDB ID

1.5 1.3 1.3

1XI1 1XHX 1XHZ

Orthorhombic

(P212121: a ⫽ 95.3 A˚, b ⫽ 149.9 A˚, c ⫽ 199.0 A˚)

Apo

0.9760

(dT)5

0.9760

50.0–2.35 9.8 (2.43–2.35) 50.0–2.7 9.4 (2.8–2.7)

Phasing Powerc Unique Reflections Centric Acentric Res. Range (A˚)

Refinement

(dT)5(monoclinic) Apo (orthorhombic) (dT)5(orthorhombic)

Resolution (A˚)

Copies in AU

Rcrystd (%)

Rfreed

Rmsd Bond (%) Length (A˚)

20.0–2.20 20.0–2.35 20.0–2.70

2 4 4

24.3 20.6 21.9

27.7 25.7 26.8

0.010 0.006 0.007

Rmerge is ⌺j|Ij ⫺ ⬍I⬎|, where Ij is the intensity of an individual reflection and ⬍I⬎ is the mean intensity for multiply recorded reflections. I/␴ is the mean of intensity divided by the standard deviation. c Phasing power is the RMS isomorphous difference divided by the RMS lack of closure. d Rcryst is ⌺||Fo| ⫺ |Fc||/⌺|Fo|, where Fo is an observed amplitude and Fc a calculated amplitude; Rfree is the same statistic calculated over a subset of the data (10%) that has not been used for refinement. a

b

catalytic carboxylates to be D12, E14, D66, and D169 (Bernad et al., 1989; Soengas et al., 1992; Esteban et al., 1994). Mutation of any one of these residues to alanine lowers exonucleolytic activity by 105. Mutation of K143 or Y165 also negatively affects catalysis without significantly changing the affinity of polymerase for ssDNA (de Vega et al., 1997; Soengas et al., 1992). These two residues face away from the active site in the φ29 DNA polymerase structures. One or both of the corresponding residues have also been observed in similar, presumably inactive, conformations in a number of B family polymerase crystal structures (Wang et al., 1996; Hopfner et al., 1999; Shamoo and Steitz, 1999; Franklin et al., 2001). In RB69 DNA polymerase, the conformation in which the tyrosine is directed away from the active site is stabilized by the formation of a hydrogen bond between Y323 and the amide group of Q171. Interestingly, the mutation Q171A increases kexo 5-fold

(Wang et al., 2004). This suggests that the ease with which this tyrosine can access alternate conformations may help to fine tune the level of exonuclease activity in the B family polymerases (Wang et al., 2004). The structure of φ29 DNA polymerase bound to (dT)5 permits the identification of residues that contact ssDNA in the exonuclease active site (Figure 2). A number of mutants with impaired ssDNA binding have been described in previous studies, and the structure confirms that T15, N62, and F65 directly contact ssDNA (de Vega et al., 1996, 1998). However, the negative effects of mutating Y59, F69, and S122 on ssDNA binding (de Vega et al., 1998, 2000) may be caused by disruption of the tertiary structure of the exonuclease domain since these residues are buried in the protein interior. Similarly, the inhibition of ssDNA binding caused by mutation of residue H61 (de Vega et al., 2000) may also be indirect since its imidazole moiety is directed away from DNA and

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Figure 2. Interactions of ssDNA with the Exonuclease Site (A) For clarity, the polymerase domain and two 5⬘ bases are omitted; a segment (residues 560–575) of the thumb backbone is shown as a green ribbon. The side chains of residues T15, N62, F65, Y148, P129 (in yellow), and L567 (green stick representation) contact the DNA. Metal ion A is in position to coordinate the pro-S nonbridging oxygen of the scissile phosphate, E14, and D169 (the position of metal ion A was determined as described in the Experimental Procedures). The DNA residues are labeled T3–T5. (B) A difference electron density map calculated at 2.7 A˚ between the cocrystallized ssDNA complex and the native protein. The map is calculated with phases from the native structure and contoured at 2.5␴ to provide an unbiased view of the electron density corresponding to bound DNA. The structure of the ssDNA complex is superimposed, with the exonuclease domain shown in red and the thumb subdomain in green. The DNA residues are labeled T1–T5.

forms hydrogen bonds with both S122 and F128. Two residues not previously identified that appear to be making important interactions with the ssDNA substrate are Y148, which stacks on the 3⬘ terminal base, and a residue from the thumb domain, L567, that lies between and interacts with the two 3⬘ terminal bases in the exonuclease active site. This latter interaction may explain why deletion of residues 563–575 in φ29 DNA polymerase reduces exonuclease activity on ssDNA substrate to below 1% of wild-type levels (Truniger et al., 2004a). TP mimics the DNA primer in the polymerase domain during the initial rounds of nucleotide incorporation, and we can ask whether it must also mimic the primer in the exonuclease domain during the editing of these nucleotides. The structure of polymerase complexed with ssDNA shows that the exonuclease domain contacts only the three 3⬘ terminal residues of DNA (T3–T5 in Figure 2). Biochemical studies show that while the TPdAMP bond cannot be hydrolyzed, a dinucleotide or longer attached to TP can be edited (Esteban et al., 1993). Taken together, these observations suggest that during editing, TP might only have to mimic T3. This requirement might be easily met since the ssDNA:polymerase structure also shows that specific hydrogen bonds are formed between the exonuclease domain and the backbone atoms of T5 and T4, but not T3. Palm, Fingers, and Thumb Subdomains The polymerase domain of φ29 DNA polymerase, like other polymerases, can be subdivided into palm, fingers, and thumb subdomains by analogy to a right hand (Figure 1). The palm subdomains of all DNA polymerases of known structure, except pol ␤, are structurally homologous and contain the conserved, catalytic metal ion binding carboxylates of the active site (Steitz et al., 1994). The DNA from the structure of the RB69 ternary complex (Franklin et al., 2001) can be homology modeled onto the structure of the apo φ29 polymerase by aligning

the palm subdomains of φ29 and RB69 DNA polymerases. These palm subdomains superimpose with an rmsd of 1.3 A˚ based on 107 pairs of C␣ atoms (φ29 DNA polymerase residues: 226–235, 238–261, 425–464, 481–505, 522–529; Figure 3). DNA homology modeled in this fashion is complementary with the surface of φ29 DNA polymerase. There is no overlap of duplex DNA with the protein main chain, though some clashes occur between DNA and side chains in the polymerase thumb subdomain. Previous examples suggest that DNA modeled in this manner, using the structure of a complex with a homologous polymerase, should be accurate to an rmsd of 1–3 A˚, calculated over the nascent and two adjacent base pairs. This range of rmsd values was obtained by comparing the coordinates of homologymodeled DNA with those that were experimentally determined between five pairs of polymerases from different families (Zhou et al., 2001). Finally, the position of the modeled DNA is consistent with the locations of the catalytic carboxylates (Figure 1; D249 and D458) (Blasco et al., 1993; Bernad et al., 1990), the steric gate residue that distinguishes ribo- from deoxyribonucleotides (Y254 [Bonnin et al., 1999]), and some residues implicated in binding DNA (K498, Y500 [mutational data reviewed by Blanco and Salas, 1996]). The finger subdomain is in an open conformation in the present φ29 DNA polymerase structures, as would be expected in the absence of substrates bound at the polymerase active site. With the exception of the Y family of lesion bypass DNA polymerases (Zhou et al., 2001; Ling et al., 2001), the closed conformation of the fingers subdomain has been observed only when the incoming dNTP correctly base pairs with the primed template DNA (Pelletier et al., 1994; Doublie et al., 1998; Huang et al., 1998; Kiefer et al., 1998; Li et al., 1998; Franklin et al., 2001). The fingers subdomain of φ29 DNA polymerase, like those in other B family polymerases, contains a pair of antiparallel ␣ helices. A 19⬚ rotation is required to bring them into alignment with the closed conformation

φ29 DNA Polymerase Structure 613

Figure 3. Homology Modeling of DNA from the Structure of a RB69 DNA Polymerase Ternary Complex onto the Structure of φ29 DNA Polymerase (A) A superposition of the RB69 DNA polymerase (Franklin et al., 2001) and φ29 DNA polymerase palms. The catalytic carboxylates are shown in space-filling representation. (B) The DNA from the RB69 ternary complex (Franklin et al., 2001) modeled onto the φ29 DNA polymerase structure using the superposition shown in (A) without any further adjustment. The positions of the modeled primer (gray), template (black), and incoming nucleotide (yellow, space-filling spheres) are indicated; the polymerase is colored as in Figure 1.

of the ternary structure of RB69 DNA polymerase. This modeled, closed conformation is consistent with mutational studies indicating that K371, K379, and K383 contact the triphosphate moiety of the incoming dNTP (Truniger et al., 2002, 2004b; Saturno et al., 1997). The thumb subdomain of φ29 DNA polymerase has an unusual structure (Figure 1). Unlike the thumb subdomains of other polymerases, it is small (45 amino acids) and has very little helical character. The subdomain is made of a loop (530–534), followed by a connector containing a short helix as well as irregular secondary structure (535–547), and ends with a long ␤-turn-␤ element (residues 548–575). Interactions between modeled DNA and the thumb are mediated by the loop and ␤-turn-␤ element (Figure 3B). These interactions seem plausible for two reasons. First, they place electrostatically positive regions of protein in contact with DNA. Second, deletion of the C-terminal 13 residues dramatically affects affinity for doublestrand DNA, presumably by destroying the extended ␤-turn-␤ structure, and converts φ29 DNA polymerase into a distributive enzyme (Truniger et al., 2004a). Terminal Protein Regions 1 and 2 The TPR1 insertion of φ29 DNA polymerase was originally proposed, on the basis of amino acid sequence comparisons, to extend from residue 302 to 358 (Dufour et al., 2000). However, examination of the structure of φ29 DNA polymerase shows that TPR1 extends from 261 to 358 (Figures 1 and 4A). With these additional residues, TPR1 forms a well-defined subdomain of mixed ␣ and ␤ structure extending from residue 261 to 358 (Figure 4A). A search of the structural database using the program DALI (Holm and Sander, 1993) yielded no structural match with a Z score higher than 2.9 (the 2.9

match was to a functionally unrelated, thiol proteinase; PDB code 1CLQ). Thus TPR1 appears to have a novel domain structure without significant similarity to existing structures. TPR1 lies at the edge of the palm subdomain, abutting the region where modeled upstream duplex DNA is bound by the polymerase. This is consistent with its being able to directly contact both TP and DNA. Three residues have been mutated within TPR1: K305, Y315, and D332. Mutations at position 305 and 315 impair the ability of φ29 DNA polymerase to use TP as a primer and also affect the rate of exonucleolysis of primer:template substrate (Dufour et al., 2003). Our structures suggest that interactions between these residues and substrate DNA are plausible since residues 302–316 form a ␤-turn-␤ structure that contacts substrate DNA in the homologymodeled complex (Figures 3B, 4A, and 5A). However, the crystallographic evidence is not definitive since this ␤-turn-␤ structure has high temperature factors and assumes different orientations in the six crystallographically independent copies of polymerase in our crystals. The highly conserved residue D332, on the other hand, seems less likely to directly interact with TP since it forms a partially buried salt bridge with R438. The mutation D332Y (Dufour et al., 2000) could thus affect the orientation of R438 of the palm subdomain as well as alter the interface between TPR1 and palm. Consequently, even though the mutation of D332 affects protein priming (Dufour et al., 2000), this phenotype may not arise from a direct contact between this residue and terminal protein. TPR2 in φ29 DNA polymerase bears a striking resemblance to the specificity loop of the pol I family T7 RNA polymerase (T7 RNAP). Superposition of the two polymerases using 56 pairs of C␣ atoms in their conserved

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Figure 4. Structures of TPR1 and TPR2, Domains that Are Specific to Protein-Primed DNA Polymerases (A) TPR1 forms a compact domain. This region is an insertion between the palm and the fingers subdomains. The motif, identified on the basis of sequence analysis (residues 302– 358, gold), can be extended to include residues 261–301 as well (brown), thereby forming a subdomain with no homology to the palm subdomains of other B family polymerases. (B) Structural analogy between TPR2 (cyan) and the specificity loop (gold) of T7 RNA polymerase. The fragments of both palms used for superposition are colored in pink (φ29 DNA polymerase) and gray (T7 RNA polymerase). The atoms of the residues containing the catalytic carboxylates are shown as space-filling spheres.

palm regions (with an rmsd of 1.9 A˚) shows that not only do TPR2 and the specificity loop both consist of long ␤-turn-␤ pairs of similar conformation, but they both also emerge from the same regions of their palm subdomains (Figure 4B). The specificity loop of T7 RNAP has been shown to serve at least two different functions: during initiation it contacts upstream promoter DNA, and during elongation it forms part of the tunnel through which the newly synthesized RNA transcript passes (Cheetham et al., 1999; Yin and Steitz, 2002). However, φ29 DNA polymerase does not perform analogous functions. Although it recognizes a specific sequence at the origin of replication, this origin is at the end of a linear genome and there is no nucleic acid upstream of it. Instead, TPR2 probably plays a critical role in contacting downstream template DNA, as discussed below. It is thus currently unclear whether the structural similarity between TPR2 and the specificity loop reflects any underlying functional similarity or evolutionary relationship. Structural Basis of Strand Displacement and Processivity The strand displacement activity and processivity of replicative polymerases is usually enhanced by accessory factors that topologically surround their DNA substrates. Thus hexameric ring helicases unwind dsDNA by threading only a single strand of DNA through their central pores (Patel and Picha, 2000). Similarly, clamp proteins associate with polymerases and increase their processivity by encircling the product upstream dsDNA (Kong et al., 1992). An examination of the three electropositive paths leading into the active site of φ29 DNA polymerase suggests that it may use the same topological mechanisms of threading ssDNA and surrounding dsDNA as helicases and sliding clamps, respectively, thereby giving it intrinsic strand displacement activity and processivity in the absence of these accessory factors (Figures 5 and 6). Residues from the exonuclease domain as well as from the palm, fingers, and thumb subdomains enclose the pathway through which dNTPs presumably enter and pyrophosphate leaves the active site (Figure 6). Functionally, this large entrance appears to be analogous to tunnels observed in other B family polymerases (Franklin et al., 2001) and RNA-dependent RNA polymerases (Tao et al., 2002) and to the “secondary channels” of multisubunit RNA polymerases (Zhang et al., 1999).

Residues from the palm, thumb, TPR1, and TPR2 subdomains and from the exonuclease domain surround a second opening into the φ29 DNA polymerase active that encircles the homology-modeled upstream duplex DNA (Figures 5A and 6). The ␤ turn at the tip of the thumb contacts the tip of TPR2 to form an arch whose structure is similar in all six of the crystallographically independent copies of the polymerase. Assuming that any movement of the thumb accompanying DNA binding does not abolish the contact of the thumb with the exonuclease domain and TPR2, this region of φ29 DNA polymerase can form an “upstream duplex tunnel” capable of encircling DNA and enhancing processivity in a fashion analogous to the sliding clamp proteins in other replisomes. The hypothesis that encirclement confers processivity is consistent with the observation that the deletion of its C-terminal 13 residues makes φ29 DNA polymerase activity distributive (Truniger et al., 2004a), and could be further tested through site directed mutagenesis. For example, G563, G564, and C22 form part of the interface between the thumb and the exonuclease domain in the current crystal structures, and a disulfide link might be engineered by mutation of either G563 or G564 to cysteine. Mutant polymerases able to initiate synthesis only close to the termini of linear DNA, or with increased processivity, would lend support to the hypothesis. We refer to the third path into the active site of φ29 DNA polymerase as the downstream template tunnel since homology-modeled downstream template DNA passes through it (Figures 5B, 5C, and 6). Residues from TPR2, the exonuclease domain, and the fingers and palm subdomains form this tunnel. It is less than 10 A˚ long and approximately 10 A˚ in diameter. Encirclement of the DNA by this tunnel may also enhance processivity, as suggested for the upstream duplex tunnel. Additionally, the narrow dimensions of this tunnel surrounding the downstream template preclude the passage of double-stranded DNA through it. As a consequence, the template and nontemplate strands of DNA would have to separate before the template could enter the tunnel, providing a structural basis for the strand displacement activity of φ29 DNA polymerase. In fact, deletion of TPR2 strongly impairs processivity of the mutant DNA polymerase (I. Rodrı´guez, J.M.L., L.B., M.S., and M.d.V., unpublished data). The steric separation of downstream template and nontemplate strands by polymerases has been ob-

φ29 DNA Polymerase Structure 615

Figure 5. Structural Basis of Processivity and Strand Displacement (A and B) Homology-modeled DNA from the RB69 DNA polymerase ternary complex is shown in the context of a space-filling representation of φ29 DNA polymerase in two different orientations. The polymerase is colored as in Figure 1: exonuclease, red; palm, pink; TPR1, gold; fingers, blue; TPR2, cyan; thumb, green. The primer strand of the DNA is colored gray, and the template is colored yellow. The orientation in (A) is similar to that in Figure 1 and shows topological encirclement of modeled upstream duplex product DNA by the thumb, palm and TPR2. The polymerase orientation in (B) is rotated approximately 75⬚ from (A) and shows modeled downstream template passing through a narrow tunnel made by the exonuclease domain, palm subdomain, and TPR2 before entering the polymerase active site. (C) An electrostatic surface representation of the polymerase. This view shows that positively charged (blue) protein surface would

served before. In the pol I family, residues from a conserved ␣ helix lie between the strands, requiring them to separate (Yin and Steitz, 2002). Even closer structural precedents to the mechanism of strand separation used by φ29 DNA polymerase may exist in the cases of φ6 and reovirus RNA-directed RNA polymerases (Butcher et al., 2001; Tao et al., 2002). In both of these polymerases, downstream template passes through a tunnel before entering the active site, though the secondary structure elements that form the tunnels in these polymerases are not homologous to those of φ29 DNA polymerase. In φ6 RNA polymerase, there is, in addition to a template tunnel, a positive surface where nontemplate RNA is proposed to bind. No clear binding site for the nontemplate strand is evident in the φ29 structures, and no plausible path for the nontemplate strand can be inferred. The structure does not explain how mutation of the exonuclease active site residues impairs strand displacement activity in in vitro M13 rolling circle replication assays (Soengas et al., 1992; Esteban et al., 1994). Though the downstream template tunnel forms a closed ring in all of the crystallographically independent structures of φ29 DNA polymerase, the protein must be able to open the tunnel because φ29 DNA polymerase is capable of replicating circular ssDNA templates. An open conformation would be required to “thread” the template into the tunnel in such a case. In vivo, a special circumstance also arises when replicating the ends of the φ29 genome. The terminal base of the template is covalently linked to TP, and the tunnel in its current conformation appears unlikely to allow such a large substrate to pass through it to enter the active site. Furthermore, following the synthesis of the last base, dissociation of the polymerase from the replicated genome requires an opened tunnel. A plausible mechanism for opening would involve the peeling away of the extended ␤-turn-␤ structure of TPR2 from its interactions with the exonuclease domain and thumb subdomain. Our structural and modeling studies of φ29 DNA polymerase have suggested that the intrinsic strand displacement and processivity of this enzyme can be explained through topological mechanisms which resemble those of helicases and clamp proteins in other replication systems. Structures of the polymerase complexed with DNA are required to test the validity of this suggestion and to present a more detailed view of the interactions between the DNA polymerase and nucleic acids. Structural studies are also needed to illuminate how φ29 DNA polymerase interacts with TP both during the initiation phase of DNA synthesis, when TP acts as a primer, as well as during the termination phase, when the polymerase encounters TP covalently linked to the 5⬘ end of a template strand. Experimental Procedures Protein Purification and Crystallization Exonuclease-deficient (D12A/D66A) φ29 DNA polymerase was expressed and purified as described elsewhere (La´zaro et al., 1995).

contact downstream template. The electrostatic surface is contoured with saturating values of blue and red set at ⫾15 kT respectively.

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Figure 6. Paths Leading to the Active Site of φ29 DNA Polymerase (A) A surface representation of the polymerase with homology-modeled DNA (primer, red; template, yellow; incoming dNTP, space-filling purple) sliced into two halves to show three paths leading into the active site. A narrow tunnel allows the modeled uncopied downstream template into the active site while a large pore provides a path for incoming dNTP. Modeled upstream product duplex exits from the polymerase active site through a tunnel of intermediate dimensions. (B) Schematic representation of φ29 DNA polymerase with DNA substrate. The protein is diagramed in two levels. The upper level contains the exonucleolytic domain, TPR2 subdomain, and thumb subdomain (outlined in red, cyan, and green, respectively). The rest of the protein is indicated in gray. An asterisk marks the polymerase active site position.

Selenomethionyl-labeled polymerase was obtained by growing B834 (DE3) cells in media supplemented with all amino acids except methionine, which was replaced with selenomethionine. Purified polymerase was precipitated using ammonium sulfate and stored at ⫺80⬚C. Protein was resuspended in 25 mM Tris-HCl (pH 7.5), 250 mM NaCl prior to crystallization trials. Crystals were grown by vapor diffusion. Standard conditions for obtaining monoclinic crystals were 5.5–11 mg/ml protein stock mixed 1:1 with a well solution of 200 mM magnesium acetate, 100 mM Tris-HCl (pH 7.5), and 10%– 20% PEG 8000 (w/v) at 20⬚C. Crystals typically appeared overnight. Examination under a microscope showed that over 95% of these crystals contained multiple lattices. Vapor diffusion experiments at 4⬚C under essentially the same conditions yielded the orthorhombic crystal form as well as monoclinic crystals. The orthorhombic crystals appeared after 1 week and were much less reproducible than the monoclinic crystals. The complex with ssDNA was prepared by soaking crystals in millimolar concentrations of (dT)5 overnight. Crystals were stabilized by transferring them to buffer typically containing 100 mM magnesium acetate, 40 mM ammonium sulfate, 20 mM Tris-HCl (pH 7.5), 22% PEG 8000 (w/v). They were transferred in steps of increasing concentrations of ethylene glycol to achieve a final concentration of 30% before freezing in liquid propane. Structure Determination, Refinement, and Analysis Data were integrated and scaled using the HKL suite of programs (Otwinowski and Minor, 1997). Three derivatives were instrumental in solving the structure of the monoclinic crystal form through a combination of MIR and MAD phasing (Table 1). Crystals were incu-

bated for 2 hr in solutions containing either (1) 25 ␮M mercury acetate and 20 mM trimethyl lead acetate (“Hg”) or (2) 20 mM trimethyl lead acetate (“Pb”). Crystals were also grown using protein containing selenomethionine (“Se”). Diffraction data from these derivatives as well as from native protein crystals “1” and “2” were corrected computationally to account for a lattice translocation defect (J.W., S.K., A.J.B., and T.A.S., unpublished data). Phases were refined and calculated using MLPHARE (CCP4, 1994) and combined by SIGMAA weighting (CCP4, 1994). Two-fold noncrystallographic symmetry averaging and solvent flattening using the program RESOLVE (Terwilliger, 2000) yielded interpretable maps. The structure of the orthorhombic crystal form was determined by molecular replacement using the structure of a polymerase monomer from the monoclinic crystal form as a search model in the program MOLREP (Vagin and Teplyakov, 1997). Using DMMULTI (CCP4, 1994), averaging between the four copies within an asymmetric unit of the orthorhombic form, as well as crosscrystal averaging with the monoclinic form yielded interpretable maps. The models were built using the program O (Jones et al., 1991). Refinement was performed in CNS (Brunger et al., 1998); tight NCS restraints were maintained during the initial stages of refinement. A monoclinic crystal stabilized with 1 mM (dT)5 and 100 mM zinc acetate was used to determine the location of metal ions in the exonuclease active site. A 5␴ anomalous difference Fourier peak indicated the position of metal ion A. No density corresponding to metal ion B was observed, presumably because of the inactivating D12A and D66A mutations in our polymerase. The position of the peak was superimposed, by least squares over all C␣ atoms in the exonuclease domain, onto the orthorhombic crystal structure shown in Figure 2.

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Figures 1, 2B, 3, and 4 were made using RIBBONS (Carson, 1991), and Figures 2A, 5A, 5B, and 6A were made using Pymol (http:// www.pymol.org). Electrostatic surfaces for Figure 5 were calculated using GRASP (Nicholls et al., 1991) and imported into MOLSCRIPT (Kraulis, 1991). RIBBONS and MOLSCRIPT figures were rendered using POV-RAY (http://www.povray.org). Acknowledgments We thank the staff at NSLS beamlines X25, X12B, X12C, and X26C, especially Michael Becker and Dieter Schneider; at APS beamlines ID19 and NECAT; at CHESS beamlines A1 and F1; and at ALS beamlines 5.0.2, 8.2.1, and 8.3.1. We also thank Catherine Joyce and Janice Pata for critical reading of the manuscript, members of the Steitz laboratory for help with data collection and useful discussions, and the staff of the CSB core facility at Yale. This work was funded in part by grant R01GM57510 from the National Institutes for Health to T.A.S., by grants BMC2002-03818 from the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica and 2R01 GM27242-24 from the National Institutes for Health to M.S., and by an institutional grant from Fundacio´n Ramo´n Areces to the Centro de Biologı´a Molecular “Severo Ochoa.” Received: July 21, 2004 Revised: September 21, 2004 Accepted: October 6, 2004 Published: November 18, 2004 References Beese, L.S., and Steitz, T.A. (1991). Structural basis for the 3⬘-5⬘ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25–33. Bernad, A., Blanco, L., La´zaro, J.M., Martı´n, G., and Salas, M. (1989). A conserved 3⬘-5⬘ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59, 219–228. Bernad, A., La´zaro, J.M., Salas, M., and Blanco, L. (1990). The highly conserved amino-acid sequence motif Tyr-Gly-Asp; Thr-Asp-Ser in ␣-like DNA polymerases is required by phage φ29 DNA polymerase for protein-primed initiation and polymerization. Proc. Natl. Acad. Sci. USA 87, 4610–4614. Blanco, L., and Salas, M. (1984). Characterization and purification of a phage phi29-encoded DNA polymerase required for initiation of replication. Proc. Natl. Acad. Sci. USA 81, 5325–5329. Blanco, L., and Salas, M. (1985). Replication of phi29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi29 DNA. Proc. Natl. Acad. Sci. USA 82, 6404–6408. Blanco, L., and Salas, M. (1996). Relating structure to function in φ29 DNA polymerase. J. Biol. Chem. 271, 8509–8512. Blanco, L., Prieto, I., Gutie´rrez, J., Bernad, A., La´zaro, J.M., Hermoso, J.M., and Salas, M. (1987). Effect of NH4⫹ ions on phi29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase. J. Virol. 12, 3983–3991.

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Accession Numbers

Rodriguez, A.C., Park, H.W., Mao, C., and Beese, L.S. (2000). Crystal structure of a pol ␣ family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7. J. Mol. Biol. 299, 447–462.

Coordinates and structure factors for the monoclinic, apo orthorhombic, and (dT)5 bound orthorhombic structures of bacteriophage φ29 DNA polymerase were deposited in the PDB under accession codes 1XI1, 1XHX, and 1XHZ.

Salas, M. (1991). Protein-priming of DNA replication. Annu. Rev. Biochem. 60, 39–71.

Note Added in Proof

Salas, M., Mellado, R.P., Vin˜uela, E., and Sogo, J.M. (1978). Characterization of a protein covalently linked to the 5⬘ termini of the DNA of Bacillus subtilis phage φ29. J. Mol. Biol. 119, 269–291. Salas, M., Miller, J.T., Leis, J., and DePamphilis, M.L. (1996). Mechanism for priming DNA synthesis. In DNA Replication in Eukaryotic Cells, M.L. DePamphilis, ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp.131–176.

The data referred to throughout as J.W., S.K., A.J.B., and T.A.S., unpublished data, are now in press. Wang, J., Kamtekar, S., Berman, A.J., and Steitz, T.A. (2004). Correction of X-ray intensities from single crystals containing lattice translocation defect. Acta Crystallogr. D, in press.