Structural Basis for Promutagenicity of 8-Halogenated Guanine*

Background: 8-Halogenated guanine is a promutagenic lesion that promotes insertion of guanine opposite the lesion during DNA replication. Results: 8-Bromoguanine forms Hoogsteen base pairing with G and Watson-Crick base pairing with C in the active site of polβ. Conclusion: 8-Bromoguanine is well accommodated in the nascent base pair binding pocket of polβ. Significance: Our structural studies provide insights into potential G to C mutations. 8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase β (polβ) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polβ with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polβ in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polβ binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polβ. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.

G and C opposite templating BrG in vitro, albeit with a ϳ100-fold reduced insertion efficiency compared with insertion of C opposite G (13). Pol␤ is a DNA repair enzyme that fills short nucleotide gaps in DNA produced during base-excision DNA repair pathway (15). This enzyme belongs to X-family DNA polymerase and lacks an intrinsic proofreading 3Ј 3 5Ј exonuclease activity. The protein contains an N-terminal lyase domain (8 kDa) and a C-terminal polymerase domain (31 kDa). The polymerase domain can be further divided into thumb, fingers, and palm subdomains typically observed in DNA polymerases.
Whereas several structures of haloG-containing nucleic acids have been published (16,17), its protein-bound structure has not been reported. Here, we report seven x-ray structures of pol␤ bound to DNA containing BrG at varying stages of DNA replication. We determined a gapped binary complex structure of pol␤ bound to templating BrG, and two ternary complex structures of pol␤ with incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. In addition, we determined four binary complex structures of pol␤ with BrG⅐C or BrG⅐G at varying positions of the enzyme active site. These x-ray structures provide structural basis for misincorporation of G opposite BrG by pol␤ and insights into potential haloGinduced G to C mutation.

EXPERIMENTAL PROCEDURES
DNA Sequences-Oligonucleotides were purchased from Integrated DNA Technologies (IDT) or Midland Certified Reagent Co. (Midland, TX). They were purified by the manufacturer, and their sequences were confirmed by MALDI-TOF mass spectrometry. DNA substrates used for crystallographic studies consisted of a 16-mer template, a complementary 10-mer primer, and 5-mer downstream oligonucleotides (18). The template DNA sequence used for crystallization was 5Ј-CCGAC[BrG]TCGCATCAGC-3Ј. The upstream primer sequence was 5Ј-GCTGATGCGA-3Ј. The downstream oligonucleotide sequence was 5Ј-GTCGG-3Ј, and the 5Ј terminus was phosphorylated. The DNA sequence almost identical to a published ternary complex structure (PDB ID 1BPY) was used to minimize sequence-dependent structural differences (19). The oligonucleotides were mixed and annealed to give a 1 mM mixture of gapped DNA as described (19).
Protein Expression and Purification-Pol␤ was expressed and purified from Escherichia coli with minor modifications of the method described previously (19,20). The human polymerase ␤ gene cloned into pET-30 (Novagen) was transformed into Rosetta2 (DE3) cells. Bacteria were grown in LB medium supplemented with 30 mg/liter kanamycin at 37°C to an A 600 ϭ 0.6, and cooled to 28°C for 30 min. Then the protein was induced by the addition of 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside. The culture was grown overnight at 28°C, harvested by centrifugation (8000 ϫ g for 10 min), resuspended in a lysis buffer (20 mM sodium phosphate, pH 7.5, 300 mM NaCl, 1 mM PMSF), and lysed by sonication for 2 min. The suspension was spun at 10,000 ϫ g for 15 min, and a clear supernatant was loaded onto a HisTrap column (GE Healthcare) pre-equilibrated with lysis buffer and eluted by the gradient of 250 mM imidazole. After pooling the fractions containing the protein and extensive buffer exchange with 50 mM Tris, pH 7.5, 2 mM DTT, and 100 mM NaCl, the concentrate was applied to Mono S cation exchange column (GE Healthcare). Then the His tag was cleaved by incubating a mixture of factor Xa and thrombin overnight at 4°C. To further separate from minor contaminants, human polymerase ␤ was purified by size exclusion chromatography on a Superdex 75 column (GE Healthcare) preequilibrated with 20 mM Tris-HCl, pH 8.0, 1 mM DTT, 200 mM NaCl. After purification, pol␤ was buffer-exchanged, concentrated to 20 mg/liter, and stored at Ϫ80°C as described.
Protein-DNA Co-crystallization-The binary pol␤ complex containing templating BrG in a single-nucleotide gapped DNA was prepared under conditions similar to those described previously (19). Pol␤ was complexed with a single-nucleotide gapped DNA containing a 16-mer template (5Ј-CCGAC[BrG]-GCGCATCAGC-3Ј), a complementary 10-mer primer (5Ј-GCTGATGCGC-3Ј), and a 5-mer downstream oligonucleotide (5Ј-pGTCGG-3Ј). The resulting pol␤-DNA complex was used to obtain binary and ternary complex crystals in the absence or presence of an incoming nucleotide, respectively. The ternary pol␤-DNA complex co-crystals with nonhydrolyzable dGTP or dCTP analog paired with templating BrG in a single-nucleotide gap at the active site were grown in a solution containing 50 mM imidazole, pH 7.5, 14 -23% PEG3400, and 350 mM sodium acetate as described previously (21). Crystals were cryo-protected in mother liquor supplemented with 12% ethylene glycol and were flash-frozen in liquid nitrogen.
Data Collection and Refinement-Diffraction data were collected at 100 K using either a Rigaku MicroMax-007 HF microfocus x-ray generator with R-Axis IVϩϩ imaging plate area detector or the beamline 5.0.3 Advanced Light Source at Berkeley Center for Structural Biology. All diffraction data were processed using HKL 2000 (22). The structures of the binary pol␤ complex with templating BrG in a single-nucleotide gapped DNA and the ternary complex of pol␤ with templating BrG paired with dGTP or dCTP analog were solved by molecular replacement with pol␤ with a single-nucleotide gapped DNA  (PDB code 1BPX) as the search model. The model was built using COOT and refined using PHENIX software (23,24). Mol-Probity was used to make Ramachandran plots (25).

Structure of a Single-nucleotide Gapped Binary Complex of
Pol␤ with Templating BrG-We determined the x-ray structure of a single-nucleotide gapped binary complex of pol␤ bound to BrG-containing DNA. The structure of the gapped binary complex (PDB ID 4M2Y, see Table 1 for refinement statistics), refined to 2.3 Å resolution, is very similar to that of a published gapped binary structure (PDB ID 1BPX, r.m.s.d. ϭ 0.689 Å) ( Fig. 2A) (19). Protein is in an open conformation, with ␣-helix N containing Asn-279 and Arg-283, the minor groove recognition motifs, being located ϳ10 Å from templating BrG.
The BrG gapped binary structure shows that, unlike G or oxoG, unpaired BrG at templating position preferentially adopts syn conformation. The syn-BrG is stabilized by an intramolecular H-bond between the N2 and the 5Ј-phosphate oxygen of BrG (Fig. 2B). In addition, an ordered water molecule stabilizes syn-BrG by bridging BrG and Tyr-271. The preferred syn conformation of BrG in the templating base position is in contrast with the previous observation that oxoG in the same position exists as a mixture of syn and anti conformers (14,18), suggesting that size of C8-substituent may govern base conformation in the templating base position. The presence of syn-BrG in DNA triggers a local conformational change at the template DNA, where the BrG lesion moved ϳ10 Å away from the position of G nucleotide residue seen in the published binary complex structure (PDB ID 1BPX) (19).
Ternary Structure of Pol␤ with Templating BrG Paired with an Incoming dCTP Analog-To elucidate structural features of pol␤ performing correct insertion opposite BrG, we obtained a pre-catalytic ternary structure of pol␤ in complex with an incoming nonhydrolyzable dCMPNPP (hereafter dCTP*) paired with templating BrG. The NH group in dNMPNPP replaces the bridging oxygen between P␣ and P␤, rendering the nucleotide analog resistant to dNMP transfer and hydrolysis. The use of nonhydrolyzable dNMPNPP thus enables the capture of ternary pol␤ structures bearing the critical coordination of 3Ј-OH of primer terminus to the catalytic metal ion (26). These nonhydrolyzable dNMPNPP nucleotides have been shown to have   active-site coordination essentially identical to that of their natural nucleotides (e.g. PDB ID 2FMP and 2FMS (26)) and have been used in structural studies of various DNA polymerases (27)(28)(29). The BrG⅐C ternary structure (PDB ID 4NLK) was refined to 2.5 Å resolution. The overall structure of the BrG⅐C ternary complex is very similar to that of published ternary structures with correct base pair, which assumes co-planar base pair conformation and closed protein conformation (PDB ID 2FMS, r.m.s.d. ϭ 0.299 Å; PDB ID 2FMP, r.m.s.d. ϭ 0.295 Å) (Fig. 3A) (26). Open-to-closed conformational activation of pol␤ for correct nucleotide incorporation typically involves the movement of ␣-helix N and the change in H-bonding interactions of Asn-279, Arg-283, and Tyr-271 with DNA (15). In the BrG⅐C ternary structure, the ␣-helix N moved ϳ10 Å from the position in the gapped binary complex to sandwich the nascent BrG⅐C base pair between the primer terminus base pair and ␣-helix N. In addition, Asn-279, Arg-283, and Tyr-271 engage in H-bonding interactions with the minor groove edges of the incoming nucleotide, templating base, and primer terminus, respectively.
The BrG⅐C ternary structure shows that the BrG⅐C base pair is well accommodated in the nascent base pair binding pocket of the enzyme. In the BrG⅐C ternary structure, BrG adopts anti conformation rather than syn conformation observed in the BrG gapped binary structure. The BrG and incoming dCTP* form co-planar a Watson-Crick base pair typically observed in structure with correct insertion (Fig. 3B) (30). The BrG⅐dCTP* is sandwiched between the primer terminus base pair and the ␣-helix N in closed conformation. The geometry of BrG⅐C base pair is essentially identical to that of G⅐C. The angles for BrG and dCTP* are 53.2°are 55.1°, respectively (Fig. 3C). The dis-tance between C1Ј(dCTP*) and C1Ј(BrG) is 10.7 Å, which is similar to that observed in structure for correct insertion.
Our structure provides insight into the slower insertion efficiency for dCTP opposite BrG (13). Distance between the 3Ј-OH of primer terminus and P␣ of incoming nucleotide (3.9 Å) is longer than that observed in correct insertion (3.4 Å), which would be suboptimal for nucleotidyl transfer. In addition, the 3Ј-OH of primer terminus is not well positioned for in-line attack on P␣ of dCTP*. Furthermore, although the BrG⅐C ternary structure adopts co-planar Watson-Crick base pairing and closed protein conformation, the coordination sphere of the catalytic metal ion is not complete. In a ternary pol␤ complex structure with correct insertion, catalytic metal ion is complexed with three Asp residues (Asp-190, -192, and -256), P␣ oxygen of the incoming nucleotide, the 3Ј-OH of primer terminus, and ordered water. In the BrG⅐C ternary structure, the ordered water molecule is not coordinated to catalytic metal ion. Combined effects of the longer 3Ј-OH-P␣(dCTP*) distance, nonideal trajectory for in-line attack, and incomplete coordination state of the catalytic metal ion would decrease the insertion efficiency for C opposite BrG.
Ternary Structure of Pol␤ with Templating BrG Paired with an Incoming dGTP Analog-To elucidate structural features of pol␤ misincorporating G opposite BrG, we solved the x-ray structure of a ternary complex of pol␤ with incoming nonhydrolyzable nucleotide analog dGTP* and templating BrG. The BrG⅐G ternary structure (PDB ID 4M47) was refined to 2.4 Å resolution (Fig. 4A).
The structure of the BrG⅐G ternary complex with mutagenic replication is very different from those of published pol␤ ternary complexes with a base pair mismatch, which typically showed staggered base pair conformation (Fig. 4A) (15,28). In the BrG⅐G ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with dGTP* in the nascent base pair binding pocket (Fig. 4B), which is reminiscent of published pol␤-oxoG⅐A ternary structure with co-planar Hoogsteen base pair (31).
Although the geometry of the nascent mismatched syn-BrG⅐anti-G base pairing deviates considerably from that of a correct nascent base pair, with a longer C1Ј-C1Ј distance (11.5 Å versus 10.5 Å) and significantly different angles (Fig. 4C), the Hoogsteen syn-BrG⅐anti-G base pair is stabilized by multiple H-bonds (Fig. 4B). The incoming dGTP* nucleotide forms two H-bonds with the Hoogsteen edge of syn-BrG. The base pair is further stabilized by an ordered water molecule that bridges O6 of both G and BrG.
Insertion of dGTP* into the enzyme active site is facilitated by the nucleotide minor-groove edge contacts to amino acid residues (Fig. 4B), with a H-bonding network significantly different from that of the published ternary complex structure (PDB ID 2FMS) (26), where Tyr-271 is H-bonded to a primer terminus base; Asn-279, an incoming nucleotide; and Arg-283, a templating base. In the BrG⅐G ternary structure, Tyr-271 is H-bonded to the minor-groove edge of N2 of dGTP*. Asn-279 and Arg-283, the minor-groove recognition motifs of pol␤ (32,33), do not engage in direct H-bondings to the minor groove of the nascent base pair. Instead, Asn-279 is H-bonded to a bridging water molecule that interacts with N3 of dGTP*. Arg-283 is not H-bonded to the templating base. Instead, it is H-bonded to the bridging water molecule. In addition to these indirect minor-groove contacts, the H-bond between 3Ј-OH of dGTP* and the backbone carbonyl oxygen of Phe-272 contributes to stabilizing the insertion (Fig. 4A).
The two metal ion-binding site of the BrG⅐G ternary structure indicates that this complex requires further conformational change to reach a catalytically competent state (Fig. 4D). Apparently, the two metal ions, the nucleotide-binding and catalytic metal ions, are inserted in the active site of the enzyme. However, whereas the coordination sphere of the nucleotidebinding metal ion is complete, that of the catalytic metal ion is not. More specifically, Asp-190, Asp-192, the P␣ oxygen of dGTP*, and a water molecule are coordinated to the catalytic metal ion, but Asp-256 and the 3Ј-OH of the primer terminus, typically involved in the completion of the coordination sphere of octahedral geometry, are not coordinated to it. Instead, Asp-256 is H-bonded to a bridging water molecule that interacts with 3Ј-OH of the primer terminus. The distance between the 3Ј-OH of the primer terminus and the P␣ of dGTP* is longer (4.4 versus ϳ3.5 Å) than the distance observed for correct insertion.
It is believed that pol␤ undergoes multiple conformational changes prior to catalyzing the nucleotidyl transfer (20,34,35), and the conformational reorganization appears to be metal coordination-dependent (32, 36 -38). Our BrG⅐G ternary structure most likely represents an intermediate conformation where binding of the two metal ions to the insertion site has occurred, yet the full conformational change required for the chemical step has not occurred. This structure also suggests that, during nucleotide insertion into the active site of the BrG is in syn conformation and is paired with incoming dGTP* through its Hoogsteen edge. Note that H-bonding interactions of Asn-279, Arg-283, and Tyr-271 in this structure are completely different from those observed in the BrG⅐C ternary structure. C, H-bonding interactions and geometry of the nascent syn-BrG⅐anti-G base pair. A water molecule that bridges O6s of G and BrG is shown as a red sphere. The distance between C1Ј(dGTP*) and C1Ј(BrG) and angles are indicated. A 2F o Ϫ F c map is contoured at 1 around BrG and dGTP*. D, close-up view of active-site metal ion binding site. Asp-256 and the 3Ј-OH of primer terminus are not liganded to the catalytic metal ion, and that distance between P␣ of dGTP* and the 3Ј-OH of primer terminus is longer than that for correct insertion (3.4 Å). Note that Na ϩ ion occupies the catalytic Mg 2ϩ ion site in this structure. Numbers in the panel indicate distances in Å. enzyme, the coordination sphere for the nucleotide-binding metal ion completes first. The coordination sphere for the catalytic metal ion completes next, with coordination of Asp-256 and/or the 3Ј-OH to the catalytic metal ion potentially taking place at the final stages of conformational change to attain optimal geometry, which would facilitate formation of the closed conformation.
Comparison of active-site structures of the gapped binary, the BrG⅐C ternary, and the BrG⅐G ternary complexes shows that protein conformation in the BrG⅐G ternary structure is between those in the gapped binary and the BrG⅐C ternary structures (Fig. 5, A-D). Although the BrG⅐G ternary complex contains two metal ions and a Hoogsteen base pair, conformation of ␣-helix N is similar to that of the gapped binary structure with open conformation (Fig. 5D), suggesting that completion of coordination spheres of the two metal ions is required for the formation of closed protein conformation.
Our syn-BrG⅐anti-G ternary structure provides insights into previously observed ϳ40-fold slower insertion of dATP opposite BrG relative to dGTP opposite BrG (13). Whereas dATP can form two H-bonds with syn-oxoG via Hoogsteen base pairing (18), it cannot form such H-bonds with syn-BrG due to mismatch between H-bond donors and acceptors (Fig. 1B); the Watson-Crick edge of dATP contains a H-bond donor and a H-bond acceptor, whereas the Hoosteen edge of BrG contains two H-bond acceptors. Therefore, formation of dATP⅐syn-BrG base pair in the nascent base pair binding pocket of the enzyme would be unfavorable.
Post-insertion Binary Structures of Pol␤ with BrG Paired with dC or dG at the N Position-To gain insights into post-insertion state of BrG⅐C and BrG⅐G, we obtained x-ray structure of binary complex of pol␤ bound to DNA containing templating BrG and primer terminus dC or dG at the N position, respectively. The BrG⅐C and BrG⅐G post-insertion structures were refined to 2.3 Å and 2.7 Å, respectively.
Interestingly, BrG⅐C at the N position of the BrG⅐C binary structure forms a staggered base pair conformation rather than co-planar conformation observed in the BrG⅐C ternary struc-ture (Fig. 6, A and B). The overall structure of BrG⅐C postinsertion complex (PDB ID 4NLN) is very similar to that of published structure with a base pair mismatch (PDB 1TV9, r.m.s.d. ϭ 0.329 Å) (39). The enzyme assumes an intermediate conformation, and BrG⅐C base pair is in staggered conformation. The BrG⅐C post-insertion structure shows an abasic site opposite templating BrG (Fig. 6B). Unlike anti-BrG in the BrG⅐C ternary structure, BrG is in the BrG⅐C binary structure is in syn conformation and does not form H-bonds with dC. The estranged dC forms two H-bonds with Arg-283 through its Watson-Crick edge (Fig. 6B).
In the BrG⅐G post-insertion structure (PDB ID 4NLZ), BrG⅐G forms staggered base pair conformation rather than coplanar conformation observed in the BrG⅐G ternary structure (Fig. 6, C and D). BrG in the BrG⅐G binary complex is in syn conformation and does not form Hoogsteen base pairing with primer terminus G. The staggered BrG⅐G base pair conformation is stabilized by an extensivestacking interaction between BrG and the primer terminus dG. The distance between BrG and dG is ϳ3.5 Å, which is similar to an average rise per base residue in B-form DNA (3.4 Å). In addition, the staggered BrG⅐G base pair is stabilized by two H-bonds formed between N2-H and N1-H of the primer terminus dG and the 5Ј-phosphate oxygen of BrG.
Post-extension Binary Structures of Pol␤ with BrG⅐C or BrG⅐G at the N-1 Position-To gain insight into base pair conformation of BrG⅐C and BrG⅐G at other positions of the enzyme active site, we determined two binary structures bearing BrG⅐C or BrG⅐G at the N-1 position. The BrG⅐C (N-1) and the BrG⅐G (N-1) binary complex structures were diffracted to 2.4 Å and 2.5 Å, respectively.
Surprisingly, in the BrG⅐C (N-1) structure (PDB ID 4NM1), protein assumes closed conformation rather than open conformation previously observed with pol␤ binary structures (Fig. 7,  A and B). The ␣-helix N moved to engage in H-bonding interactions with the primer terminus base pair. BrG and dC form a co-planar Watson-Crick base pair rather than staggered base pair seen in the BrG⅐C (N) structure. A noteworthy observation  ). B, overlay of the gapped structure (gray) with the BrG⅐C ternary structure (yellow and red). C, overlay of the BrG⅐G ternary structure (green and blue) with the BrG⅐C ternary structure (yellow and red). D, overlay of the gapped binary, BrG⅐G ternary, and BrG⅐C ternary structures.
from this closed binary structure is the presence of penta-coordinated metal ion near the primer terminus base. The metal ion is coordinated with the 5Ј-phosphate oxygen of primer terminus, monophosphate oxygen, a water molecule, Asp-190, and Asp-192. H-bondings and the base pair geometry of the BrG⅐C at the N-1 position are very similar to those of the normal Watson-Crick base pair (Fig. 7C).
In the BrG⅐G (N-1) structure (PDB ID 4NM2), protein is in open conformation (Fig. 7D). BrG is in syn conformation and forms a co-planar Hoogsteen base pair with G (Fig. 7E). As seen in the BrG⅐G ternary structure, the O6 of BrG is H-bonded to the N1 of G, and the N7 of BrG is H-bonded to the N2 of G (Fig.  7F). However, the water-mediated H-bonding between O6 of BrG and O6 of G is lacking in this structure. The C1Ј-C1Ј distance for BrG and G is ϳ1 Å longer than that for correct base pair (11.4 Å versus 10.4 Å). The angles for BrG and G are similar to those seen for BrG⅐G ternary structure.

DISCUSSION
Comparison of our BrG⅐C and BrG⅐G ternary structures with published pol␤ ternary structures suggests that the coordination state of the catalytic metal ion dictates protein conformation of pol␤ ternary complex (35)(36)(37)(38). Apparently, the coordination state of the catalytic metal ion varies among pol␤ ternary structures with different conformations. In pol␤ ternary structure with correct insertion, the catalytic metal ion adopts octahedral geometry by coordinating to the three catalytic Asp residues (Asp-190, -192, and -256), the 3Ј-OH of primer terminus, P␣ oxygen of an incoming nucleotide, and an ordered water molecule. Protein in such structure generally adopts closed conformation. In our BrG⅐C ternary structure with correct insertion, although the coordination of a water molecule to the catalytic metal ion is lacking, the complex adopts closed protein conformation, indicating that coordination of water molecule to the catalytic metal ion is not required for the conformational activation of pol␤. In pol␤ ternary structure with incorrect insertion, catalytic metal ion does not form octahedral coordination geometry and protein adopts intermediate conformation. In such a structure, Asp-256, the 3Ј-OH of primer terminus, and/or water molecule is not liganded to the catalytic metal ion, producing an incomplete coordination sphere of the catalytic metal ion. Published pol␤ ternary structure with dATP⅐dC (PDB ID 3C2L) or dATP⅐dG (PDB ID 3C2M) mismatch lacks the coordination of the primer terminus 3Ј-OH to the catalytic metal ion and shows intermediate protein conformation (28), indicating that the 3Ј-OH coordination to the catalytic metal ion is required for the conformational activation of pol␤. In our BrG⅐G ternary structure, although the complex assumes Hoogsteen base pair conformation, coordination of Asp-256, primer terminus 3Ј-OH, and water molecule to the catalytic metal ion is lacking, and the complex adopts intermediate protein conformation. Taken together, these observations indicate that the coordination state of the catalytic metal plays an important role in conformational activation of pol␤. Pol␤ appears to utilize the coordination state of catalytic metal ion as a kinetic checkpoint to discourage nucleotide misincorporation, which would be consistent with an induced-fit mechanism (19,40), where an optimal conformation for nucleotidyl transfer is allowed for correct insertion, but not for incorrect insertion.
Structural differences between the BrG⅐C (N-1) binary structure with closed protein conformation and the BrG⅐G (N-1) binary structure with open protein conformation provide new insight into post-chemistry conformational change occurring during the catalytic cycle of pol␤ (Fig. 7, B and D). Currently, whereas pre-chemistry conformational change of pol␤ is relatively well understood (19,35,36,41,42), its post-chemistry conformational change is poorly understood due largely to the scarcity of pol␤ structure with post-chemistry conformational intermediate. Comparison of reported pol␤ binary structures with the BrG⅐G (N-1) and the BrG⅐C (N-1) binary structures suggests that the metal-ion coordination observed in the BrG⅐C (N-1) binary structure plays an important role in post-chemistry conformational reorganization and prevents closed-toopen conformational inactivation (Fig. 7B). In published pol␤ binary post-insertion structures with a correct base pair, metalion coordination similar to that observed near the primer ter-minus of our BrG⅐C (N-1) binary complex is lacking, and protein adopts open conformation. In pol␤ post-insertion binary structure with A⅐C or T⅐C mismatch (31), a single Mg 2ϩ was observed, but this metal ion was not coordinated to amino acids. In our BrG⅐C (N-1) binary structure, the metal ion is coordinated to Asp-190, Asp-192, the 5Ј-phosphate oxygen of primer terminus, monophosphate oxygen, and water molecule. Interestingly, the metal ion and the monophosphate in the BrG⅐C (N-1) complex are similarly located where the nucleotide-binding metal ion and ␤-phosphate of an incoming nucleotide of the BrG⅐C ternary structure are. This suggests that the metal ion seen in the BrG⅐C (N-1) binary structure possesses coordination chemistry similar to that of the nucleotide-binding metal ion. As binding of nucleotide-binding and catalytic metal ions to the enzyme active site is believed to occur in discrete steps (30,(35)(36)(37)42), release of the metal ions from the active site could occur in a stepwise fashion (42). Our BrG⅐C (N-1) binary structure with one metal-ion coordination state and closed protein conformation suggests that, during postchemistry conformational reorganization, release of catalytic metal ion from the enzyme active site precedes that of nucleotide-binding metal ion and that the release of nucleotide-binding metal ion triggers a closed-to-open conformational inactivation. The BrG⅐C (N-1) binary structure would thus represent a close approximation of a post-chemistry conformational intermediate, where nucleotidyl transfer and catalytic metalion release have occurred, yet the release of nucleotide-binding metal ion and pyrophosphate from the enzyme active site has not occurred.
Pol␤ lacking an intrinsic proofreading exonuclease activity has shown to prevent nucleotide misincorporation by disallowing co-planar base pair conformation in the nascent base pair binding pocket (28,30,41). Mismatched base pairs such as A⅐C, T⅐C, and A⅐G have been shown to form nonplanar, staggered conformation in the enzyme active site (28,41). The formation of Watson-Crick or Hoogsteen base pair conformation observed in the BrG⅐C and the BrG⅐G ternary structures and published oxoG⅐dATP and oxoG⅐dCTP ternary structures thus indicates that some 8-modified G can be tolerated in the enzyme active site (Fig. 8, A-C). Apparently, the nascent base pair binding pocket of pol␤ is plastic enough for accommodating both Hoogsteen and Watson-Crick base pairings. To accommodate a Hoogsteen syn-BrG⅐C base pair at the N position, template DNA containing BrG shifted ϳ1 Å (Fig. 8, B and  D). To accommodate a Watson-Crick anti-BrG⅐C base pair at the N position, the 5Ј-phosphodiester backbone of BrG underwent conformational reorganization, which prevented a steric clash between the 5Ј-phosphate of BrG and the bromine moiety (Fig. 8, C and E). The similar conformational reorganization of templating base and its phosphate backbone has been observed in pol␤ ternary structure with Hoogsteen syn-oxoG⅐dATP or Watson-Crick anti-oxoG⅐dCTP base pair (14,18).
Our BrG⅐G ternary complex structure shows that the mutagenic Hoogsteen syn-BrG⅐anti-G base pairing is tolerated in the nascent base pair binding pocket of pol␤, implying that BrG lesion could potentially promote G to C mutation by utilizing the two H-bond acceptors on its Hoogsteen edge (Fig. 1B). G to C transversion mutations comprise approximately 25% of mutations found in breast cancers (43), yet only a few mutagenic lesions promoting such mutations are known (44,45), none of them utilizing the Hoosteen edge of the lesions in their mutagenic base pairings. Although further investigation will be required to evaluate potential haloG-induced mutagenesis, our studies suggest that a modified G with a bulky C8-substituent may adopt syn conformation and could facilitate G to C mutation by forming Hoogsteen base pairing using two H-bond acceptors on its Hoogsteen edge during DNA replication.