Supporting Information - PNAS · 2008. 12. 23. · Supporting Information Evans et al. 10.1073/pnas.0809989106 SI Materials and Methods Protein Expression and Purification. The polC

Supporting InformationEvans et al. 10.1073/pnas.0809989106SI Materials and MethodsProtein Expression and Purification. The polC gene encoding full-length Geobacillus kaustophilus DNA polymerase IIIC(GkaPolC) was amplified by standard PCR techniques fromgenomic DNA (90A; Bacillus Genetic Stock Center). In addi-tion, several N-terminal truncations of PolC were constructed.The resulting fragments were cloned into the T7 expressionvector pET32 (Novagen) between the NdeI and BamHI sites. Anoligonucleotide primer was used to add a C-terminal polyhisti-dine tag (SGGH8) to aid in protein purification. The exonucleasedomain (residues 425–617) was deleted and replaced with theamino acid linker Gly-Ser-Gly-Ser-Gly by ligating an N-terminalPCR fragment of PolC (residues 228–424) with a C-terminalfragment (residues 618-1444-His tag) using a common ApaI sitegenerated with the appropriate primers. Plasmids were trans-formed into BL21 (DE3) Star (Invitrogen) for expression.Cultures were grown at 37 °C until A600 � �2.0 and induced withIPTG for 1.5 h. The cells were lysed with a homogenizer (Niro),and PolC was purified by using Ni–nitrilotriacetic acid–Sepharose (Qiagen), Mono Q (GE Healthcare) and Superdex-200 (GE Healthcare) chromatography. Purified protein wasconcentrated to 10–20 mg/mL, flash frozen, and stored at�80 °C.

Protein Crystallization and Structure Solution. Oligonucleotides used incrystallization. The oligonucleotides were synthesized by IDT andpurified by the manufacturer by HPLC. The sequence of thetemplate strand was 5�-ATA ACG GTT GCC CGT CTC ACTG-3�, and the sequence of the primer strand was 5�-CAG TGAGAC GGG CAA CC-3�, where the 3� cytosine residue is a2�,3�-dideoxynucleotide. The oligonucleotides were dissolved inTE buffer, combined in equimolar ratios, heated to 95 °C,allowed to cool slowly to room temperature to facilitate anneal-ing, and were used in crystallization without further purification.Protein crystallization. GkaPolC complexes were crystallized bysitting-drop vapor diffusion. Crystals could be obtained fromcrystallants containing 200 mM lithium sulfate with a variety ofhigh-molecular mass PEGs and phosphate, citrate, or phos-phate–citrate buffers at pH 5.3–5.6 (see Table S1 for exactcrystallant used for each experiment). Crystals typically ap-peared in 1–2 days, had a trapezoidal, wedge-shaped habit, andgrew to full size of 100–200 �m in the longest dimension in 1week. Crystals were prepared for data collection by rapidlypassing them through solutions consisting of reservoir solutionplus ethylene glycol, raising the concentration of ethylene glycolin increments of 5% until a final concentration of 25% wasreached. Divalent metals and dGTP were present in all of thestabilization and cryoprotection solutions. Immediately aftertransfer to the 25% ethylene glycol cryoprotectant, crystals wereflash-frozen in liquid nitrogen.Data collection and structure solution. The structure of GkaPolC wasoriginally solved by single anomalous dispersion (SAD) phasingfrom data collected from a crystal containing selenomethionineprotein. Data were collected at beamline 5.0.2 of the AdvancedLight Source with the X-ray beam tuned to the selenium peakwavelength (0.979652 Å). The data were integrated and scaledto a resolution of 2.8 Å by using the HKL2000 software (1). SADphasing was conducted by using the program Crank (2), andphases were calculated from 23 selenium sites. The SeMet datawere combined with a 2.2-Å native dataset, and a final model wasbuilt by using ARP/wARP and multiple rounds of manual modelbuilding in Coot (3) alternated with restrained refinement by

using REFMAC5 (4). The crystals used for initial structuresolution contained the exact same GkaPolC construct and nearlyidentical DNA oligonucleotides as the structures described here,but they included a proprietary small-molecule inhibitor insteadof nucleotide triphosphate.Molecular replacement. The structures of the GkaPolC–DNA–dGTP complexes were solved by molecular replacement by usingthe protein and DNA portions of the inhibitor-bound ternarycomplex as a search model. Molecular replacement was per-formed by using the program MOLREP (5). The molecularreplacement solutions were subjected to alternating rounds ofmanual model building by using Coot (3) and restrained refine-ment by using REFMAC5 (4).

Modeling of �-Clamp and Exonuclease Domain. We first extendedthe duplex end of the PolC DNA with a straight B form DNA.The DNA in the Eco� co-crystal structure (6) [Protein DataBank (PDB) ID code 3BEP] was then aligned with the extendedPolC DNA duplex at the base pair step where the HhH motif inthe duplex binding (DB) domain of PolC was just making contactwith Eco�. We presume that the C terminus (1440–1444) thatcontains the �-binding motif (7) would be mobile in solution(and potentially all residues from Gly-1430 to end) and wouldextend away from the DB domain. In this position, the clamp andpolymerase together would span 27–28 bp of DNA duplex. Wethen superimposed Eco� that had been co-crystallized with aclamp-binding peptide from PolIV (8). The structure of theisolated exonuclease domain from Thermotoga maritima PolChas been solved (PDB ID code 2P1J) and is shown as a modelfor the probable size and insertion point for the exonucleasedomain. Relative to the deletion made in GkaPolC, this exonu-clease structure is missing both N-terminal and C-terminalresidues, making it difficult to model with any confidence theconnection to the polymerase and histidinol phosphatase (PHP)domain. The active site of the exonuclease domain is presumedto be oriented toward the DNA in the PolC structure, however.

Polymerase Activity Assays. Polymerase activity was measured ina primer extension assay by using a short oligonucleotide primer–template. Incorporation of [3H]dTTP was measured by scintil-lation proximity assay. Titrations of PolC were performed, andspecific activities were determined by using points in the linearrange. Exonuclease activity was measured by using an oligonu-cleotide primer–template with double-stranded DNA degrada-tion detected with PicoGreen fluorescence. Km

DNA was deter-mined by standard Michaelis–Menten kinetics measurements,titrating oligonucleotide in the primer–template extension assayand oligonucleotide titrations.

PolC PHP 5�–3� and 3�–5� Exonuclease Assays. The crystallizedGkaPolC has an exonuclease domain deletion. Thus, it containsa PHP domain that bears striking similarity to the DnaE PHPdomain but is incapable of catalyzing exonucleolytic cleavage bythe well-characterized exonuclease motif. This truncated form ofPolC was used to test for the existence of an exonuclease activityderiving from the PHP domain. This was compared with wild-type GkaPolC and full-length GkaPolC in which the exonucleasedomain was inactivated by a double mutation of catalytic aspar-tates. Primers were either 5� end-labeled with [�-32P]ATP or 5�or 3� end-labeled with 3�-[�-32P]dATP (PerkinElmer). Annealedoligonucleotides formed totally complementary (matched) sub-strates, or the 3� nucleotide on the primer was mismatched.

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Reactions contained 25 mM Tris�HCl (pH 8.0), 25 mM NaCl, 4mM MgCl2, 25 �M substrate, and 25 �M Tris(2-carboxyeth-yl)phosphine (TCEP). Designated assays contained Zn2� at 25�M or 1 mM and Mn2� at 1 mM. TthDnaE was prepared asdescribed in ref. 9. TthDnaE was used at 2.25 �M. Wild-type andthe crystal structure form of GkaPolC were used 0.016 and 6.8�M, respectively, and concentrations of other forms of GkaPolCwere as indicated. Reactions were carried out at 55 °C for 30 min,or at indicated times, and analyzed on 20% TBE precast gels(Invitrogen).

Matched P/T

5�-TCGAATCTGTCCTGTGTG-3�3�-AGCTTAGACAGGACACACAAGGACGACAGAGGCAAAG-5�

Mismatched P/T

5�-TCGAATCTGTCCTGTGTT-3�3�-AGCTTAGACAGGACACACAAGGACGACAGAGGCAAA

1. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscil-lation mode. Methods Enzymol 276:307–326.

2. Ness SR, de Graaff RA, Abrahams JP, Pannu NS (2004) CRANK: New methods forautomated macromolecular crystal structure solution. Structure 12:1753–1761.

3. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. ActaCrystallogr D 60:2126–2132.

4. Murshudova GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular struc-tures by the maximum-likelihood method. Acta Crystallogr D 53:240–255.

5. Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular replace-ment. J Appl Crystallogr 30:1022–1025.

6. Georgescu RE, et al. (2008) Structure of a sliding clamp on DNA. Cell 132:43–54.7. Bruck I, Georgescu RE, O’Donnell M (2005) Conserved interactions in the Staphylococ-

cus aureus DNA PolC chromosome replication machine. J Biol Chem 280:18152–18162.8. Burnouf DY, et al. (2004) Structural and biochemical analysis of sliding clamp–ligand

interactions suggest a competition between replicative and translesion DNA poly-merases. J Mol Biol 335:1187–1197.

9. Bullard JM, et al. (2002) DNA polymerase III holoenzyme from Thermus thermophilusidentification, expression, purification of components, and use to reconstitute aprocessive replicase. J Biol Chem 277:13401–13408.

10. Bailey S, Wing RA, Steitz TA (2006) The structure of T. aquaticus DNA polymerase III isdistinct from eukaryotic replicative DNA polymerases. Cell 126:893–904.

11. Lamers MH, et al. (2006) Crystal structure of the catalytic �-subunit of E. coli replicativeDNA polymerase III. Cell 126:881–892.

12. Krishna SS, Majumdar I, Grishin NV (2003) Structural classification of zinc fingers:Survey and summary. Nucleic Acids Res 31:532–550.

13. Barnes MH, Leo CJ, Brown NC (1998) DNA polymerase III of Gram-positive eubacteriais a zinc metalloprotein conserving an essential finger-like domain. Biochemistry37:15254–15260.

14. Bochkarev A, Bochkareva E, Frappier L, Edwards AM (1999) The crystal structure of thecomplex of replication protein A subunits RPA32 and RPA14 reveals a mechanism forsingle-stranded DNA binding. EMBO J 18:4498–4504.

15. Theobald DL, Mitton-Fry RM, Wuttke DS (2003) Nucleic acid recognition by OB-foldproteins. Annu Rev Biophys Biomol Struct 32:115–133.

16. Omi R, et al. (2007) Crystal structure of monofunctional histidinol phosphate phos-phatase from Thermus thermophilus HB8. Biochemistry 46:12618–12627.

17. Teplyakov A, et al. (2003) Crystal structure of the Escherichia coli YcdX protein revealsa trinuclear zinc active site. Proteins 51:315–318.

18. Stano NM, Chen J, McHenry CS (2006) A coproofreading Zn2�-dependent exonucleasewithin a bacterial replicase. Nat Struct Mol Biol 13:458–459.

19. Wing RA, Bailey S, Steitz TA (2008) Insights into the replisome from the structure of aternary complex of the DNA polymerase III �-subunit. J Mol Biol 382:859–869.



Fig. S1. Domainwise superimpositions of GkaPolC and TaqDnaE. PolC structures are colored, and TaqDnaE structures (PDB ID code 2HPI) are shown in white.Root mean square deviations for pairwise domain alignments are indicated.



Fig. S2. Zinc finger in PolC palm domain. The palm domain contains a zinc finger in which the metal ion is coordinated by 4 highly conserved cysteines (Cys-919,Cys-922, Cys-944, and Cys-947). (A) Cysteine cluster in PolC exhibiting tetrahedral metal coordination. (B) PolC exhibits a ‘‘Gag knuckle’’-type zinc finger (12). (C)Anomalous difference maps associated with the zinc finger in the palm domain of PolC. Regardless of the metal composition of the crystallization mixture([Mg2�]/[Zn2�], [Mg2�]only, or [Mn2�]/[Zn2�]), a strong anomalous difference peak is visible in the zinc finger. The contours of these maps are displayed at the5.0 � level with magenta mesh. This same feature is also present in crystals of PolC grown and frozen in the absence of additional metal salts (data not shown),indicating that [Zn2�] at this site likely copurifies with the protein. Inductively coupled plasma analysis (semiquantitative ICP; Quantitative Technologies, Inc.)of PolC purified in the presence of divalent chelators showed �0.6 mol eq of zinc and 0.4 mol eq of iron, indicating that the site might be occupied by a mixtureof zinc and iron (data not shown). This finding is consistent with atomic absorption studies by Barnes et al. (13) showing that 1 mol eq of zinc is tightly boundto PolC (i.e., copurifies with the protein in the presence of divalent chelators) but is lost upon mutation of any of the cysteine residues in the zinc finger. Theyalso reported that PolC purified in the absence of divalent chelators had �4 mol eq of zinc bound and that PolC isolated under iron-rich growth conditions had1 mol eq of iron instead of zinc. This could reflect occupancy of the PHP metal-binding site by surface-accessible metals that are stripped away by EDTA, whereasthe less accessible zinc finger metal site remains occupied by zinc or iron even in the presence of EDTA.



Fig. S3. Metal binding in the PolC PHP domain. (A and B) PHP domain �-barrel architecture and metal binding in (A) PolC [Mn2�]/[Zn2�] structure (PDB ID code2F2D) and (B) TaqDnaE (PDB ID code 2HPI). Metal coordinating residues are shown in red (His-346, His-348, Asp-355, His-380, Glu-405, Asn-743, His-745, His-620,and Cys-670 in GkaPolC). The antiparallel �-strand that is unique to PHP domains from C family DNA polymerases is shown in green. Metal coordination is similarto other structurally characterized PHP motifs such as histidinol phosphate phosphatase (16) and YcdX (17). (C–E) 2Fo � Fc maps and anomalous difference mapsfor PHP domain. Magenta contours show the anomalous difference maps at 3.0 � for the [Mg2�]/[Zn2�] structure (C) and at 5.0 � for [Mn2�]-only (D), and[Mn2�]/[Zn2�] (E). There are no anomalous difference peaks above the noise level in the [Mg2�]/[Zn2�] maps, suggesting binding of [Mg2�]. The [Mn2�]-onlystructure has 2 anomalous peaks consistent with [Mn2�] and a bound small molecule, modeled as sulfate. The [Mn2�]/[Zn2�] structure has 3 strong peaks in theanomalous difference map, modeled as 2 [Mn2�] ions and 1 [Zn2�] ion with an associated phosphate.



Fig. S4. Oligonucleotide/oligosaccharide-binding (OB) domain exhibits marked similarity to ssDNA-binding (SSB) domain. (A) Alignment of PolC OB domain(yellow) with RPA70 co-crystallized with ssDNA (magenta; rmsd 3.41 Å; PDB ID code 1JMC). (B) Alignment of PolC OB domain (yellow) with RPA32 (blue, PDBID code 1QUQ; rmsd 2.31 Å). Aromatic residues (RPA70-W361, F386) that interact with the DNA bases in the RPA70 co-crystal are shown, along with thecomparable residues in PolC (F283, F313) and RPA32 (W107, F135). Several polar residues in RPA70 (R335, S336, R339) that interact with the DNA backbone areshown, along with the comparable residues in PolC (K261, S262, R264). The polar residues are found in the �1–�2 loop implicated in OB substrate recognition(14, 15). This loop is in an extended conformation in apo-RPA70 (PDB ID code 1FGU) but wraps around the DNA backbone in the DNA co-crystal structure (picturedin A). Although the loop is somewhat shorter in PolC, a similar closing upon DNA binding could bring these polar residues in contact with the DNA. Under certainconditions, PolC ternary complexes exhibit interactions between the ssDNA template and the OB domain of a symmetry-related molecule in the crystal lattice,including stacking interactions with Phe-283 and hydrogen bonding interactions with Arg-264 (D.R.D., unpublished results). The fact that these same residuesare implicated in ssDNA recognition suggests that the crystal-packing interactions with a symmetry-related OB domain may have functional relevance.



Fig. S5. PolC sequence alignment. PolC amino acid sequences from 12 Gram� bacteria were aligned by using the ClustalW algorithm within Vector NTI(Invitrogen). Domains are indicated by the colored lines above the sequences according to the key shown. �-Helices (boxes) and �-strands (arrows) are shownoverlying the domain lines and numbered sequentially by domain. GkaPolC residues discussed in the text are identified above the secondary structure elements.Similar residues between orthologs are shown black on green, with �50% identity shown blue on cyan and 100% identity shown red on yellow. PolC orthologsequences are from G. kaustophilus (YP�147111), Bacillus subtilis (P13267), Bacillus anthracis (NP�846198), Listeria monocytogenes (CAC99398), Enterococcusfaecalis (AAM46912), Staphyloccus aureus (BAB20885), Staphyloccus epidermidis (Q8CPG6), Streptococcus pyogenes (NP�665491), Streptococcus pneumoniae(EDK67057), Clostridium tetani (Q895K2), Clostridium difficile (CAJ68162), and Thermotoga maritima (AAC80438).



Fig. S5. Continued



Fig. S5. Continued



Fig. S5. Continued



Fig. S5. Continued



Fig. S6. Enzymology of GkaPolC and TthDnaE. (A) Domains are shown for full-length PolC and for various truncation and deletion variants, including thecrystallized version (*). Polymerase primer extension and 3�–5� exonuclease activity and DNA substrate Km values are indicated (see SI Materials and Methods).(B–G) T. thermophilus DnaE (TthDnaE) exhibits 3�–5� exonuclease activity (A. Ettinger, Keystone Symposium on DNA Replication and Recombination, 2005,Keystone, CO. “Unique Characteristics of the Thermophilic DNA Polymerase III”) that resides in the PHP motif (18, 19) and was used as a control to test for thepresence of exonuclease activity associated with the PHP domain of GkaPolC. (B) 3�-5� exonuclease activity is observed in wild-type GkaPolC, which contains thewell-characterized 3�–5� exonuclease domain, which bifurcates the PHP domain. (C) TthDnaE (containing an intact PHP domain) has intrinsic 3�–5� exonucleaseactivity, but the crystal structure form of GkaPolC (which has the exonuclease insertion domain removed, thus bringing together the 2 halves of the PHP domain)lacks any exonuclease activity. (D) Analysis of exonuclease activity of TthDnaE and the crystal structure form of GkaPolC at different incubation times. (E) Effectsof different divalent ions (Mg2�, Zn2�, and Mn2�) on the activity of TthDnaE and the crystal structure form of GkaPolC. Fe2� also failed to support exonucleaseactivity (data not shown). (F) Analysis of exonuclease activity of GkaPolC with the N-terminal deletion and with the exonuclease domain present but mutatedto an inactive form. (G) Analysis of the exonuclease activity of GkaPolC without the exonuclease domain but containing the N-terminal region. In B, the primeris 5� end-labeled, and in C–E the primer is 3� end-labeled. In F and G the mismatched P/T (see SI Materials and Methods) is used as a substrate.


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Fig. S7. Structure-based alignment of PolC and DnaE. Each structural domain of GkaPolC was overlain separately with those of TaqDnaE (10) (2HPM, 2HPI) andEcoDnaE (11) (2HNH) by using PyMOL(DeLano Scientific LLC). The sequences shown were aligned primarily by secondary structure elements and secondarily byamino acid sequence conservation. The position of the GkaPolC N-terminal and exonuclease domains that were deleted in the crystallized construct are notedin the PolC sequence. Residue numbering above the sequences is that of GkaPolC. The number of the first residue of each line for all 3 orthologs is shown tothe left of the sequence. PolC domains are indicated by the colored lines above the sequences according to the key shown. PolC �-helices (boxes) and �-strands(arrows) are shown overlying the domain lines and numbered sequentially by domain. TaqDnaE structural elements are shown below the sequences and havethe published numbering (10). PolC residues discussed in the text are identified above the PolC secondary structure elements.



Fig. S7. Continued



Fig. S7. Continued



Fig. S8. Detailed view of PolC active site. (A–C) Omit maps and anomalous difference maps for the PolC active site. Fo � Fc omit maps (green contours) werecalculated for the complete PolC–DNA complex with dGTP, metals, and solvent removed from the model; maps are displayed at the 1.5 � level. Magenta contoursshow the anomalous difference maps at 3.0 � for the Mg2�/Zn2� structure and at 5.0 � for the Mn2�-containing complexes. (D) Stereoview of the PolC active siteshowing hydrogen bonds to minor groove.



Table S1. Data collection and refinement statistics

Complex High �Mg2�� and Zn2� Mn2� only Mn2� and Zn2�

Crystallization conditions 100 mM Na/K phosphate (pH 5.3), 10%PEG 8000, 200 mM lithium sulfate

100 mM citrate/citric acid (pH 5.6),10% PEG 8000, 200 mM lithiumsulfate

100 mM Na/K phosphate (pH 5.3), 14%PEG 2000-MME, 200 mM lithiumsulfate

Resolution, Å 2.4 2.5 2.5Space group C2221 C2221 C2221

Unit cell, Å a � 116.17, b � 140.79, c � 184.74 a � 115.89, b � 141.19, c � 184.74 a � 116.00, b � 139.48, c � 184.07Rsym, %* 10.1 (82.4) 9.0 (58.9) 8.7 (64.2)Ave I/�† 25.03 (1.90) 20.73 (2.24) 22.19 (2.0)Completeness, % 99.9 (100) 99.1 (91.7) 99.8 (99.0)Redundancy 5.8 (4.8) 5.9 (3.9) 4.9 (4.2)Refinement

Rcryst‡ 22.9 21.0 21.6

Rfree§ 27.3 25.5 25.4

Rms deviationsBond lengths, Å 0.0123 0.0097 0.0128Bond angles, � 1.609 1.452 1.701

Chirality 0.108 0.094 0.101

*Rsym � ( Ihkl � I�P/(Ihkl), where the average intensity I� is taken over all symmetry equivalent measurements and Ihkl is the measured intensity for any givenreflection.

†I/� is the mean reflection intensity divided by the estimated error.‡Rcryst � �FoP �PFc�/PFoP, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.§Rfree is equivalent to Rcryst but calculated for 5% of the reflections chosen at random and omitted from the refinement process.



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Supporting Information - PNAS · 2008. 12. 23. · Supporting Information Evans et al. 10.1073/pnas.0809989106 SI Materials and Methods Protein Expression and Purification. The polC