Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Oct 5.
Published in final edited form as: Biochem J. 2020 Mar 13;477(5):937–951. doi: 10.1042/BCJ20190906

Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion

Hala Ouzon-Shubeita 1,*, Caroline K Vilas 1,*, Seongmin Lee 1
PMCID: PMC7534533  NIHMSID: NIHMS1632520  PMID: 32039434

Abstract

The cisplatin-1,2-d(GpG) (Pt-GG) intrastrand cross-link is the predominant DNA lesion generated by cisplatin. Cisplatin has been shown to predominantly induce G to T mutations and Pt-GG permits significant misincorporation of dATP by human DNA polymerase β (polβ). In agreement, polβ overexpression, which is frequently observed in cancer cells, is linked to cisplatin resistance and a mutator phenotype. However, the structural basis for the misincorporation of dATP opposite Pt-GG is unknown. Here, we report the first structures of a DNA polymerase inaccurately bypassing Pt-GG. We solved two structures of polβ misincorporating dATP opposite the 5′-dG of Pt-GG in the presence of Mg2+ or Mn2+. The Mg2+-bound structure exhibits a sub-optimal conformation for catalysis, while the Mn2+-bound structure is in a catalytically more favorable semi-closed conformation. In both structures, dATP does not form a coplanar base pairing with Pt-GG. In the polβ active site, the syn-dATP opposite Pt-GG appears to be stabilized by protein templating and pi stacking interactions, which resembles the polβ-mediated dATP incorporation opposite an abasic site. Overall, our results suggest that the templating Pt-GG in the polβ active site behaves like an abasic site, promoting the insertion of dATP in a non-instructional manner.

Introduction

Cisplatin (cis-diamminedichloroplatinum(II)) is one of the most commonly employed anti-cancer drugs. Over the past four decades, cisplatin has been used to treat numerous cancers, including but not limited to ovarian, cervical, and breast cancers [1–3]. As a platinum-based chemotherapeutic agent, cisplatin exerts its cytotoxicity by targeting DNA to form mutagenic platinum-DNA (Pt-DNA) adducts. Upon cell entry, cisplatin is aquated to form a reactive platinum species. The resultant potent electrophile reacts with vulnerable nucleophilic N7 positions of purines to form platinum covalent linkages [4]. These Pt-DNA lesions generated by cisplatin include monoadducts, interstrand cross-links, and intrastrand cross-links, though the vast majority of cytotoxic lesions formed are cisplatin-1,2-d (GpG) (Pt-GG) intrastrand cross-links (Figure 1) [1,5]. The Pt-adducts formed by cisplatin inhibit essential biological processes such as replication and transcription, promoting genomic instability, cell cycle arrest, and apoptosis.

Figure 1. Cisplatin and the major cisplatin-DNA lesion.

Figure 1.

Open in a new tab

(A) Cisplatin undergoes intracellular aquation, replacing the chloride moieties to form a reactive electrophilic intermediate. (B) The intermediate platinum species attacks nucleophilic N7 atoms of adjacent purines, preferentially forming Pt-GG intrastrand cross-links, among other Pt-DNA lesions.

Despite cisplatin’s popularity and clinical efficacy, tumors often exhibit intrinsic resistance and notably, a significant fraction of tumors develop acquired resistance in response to treatment [6,7], particularly ovarian cancer [8]. The development of carboplatin provided a promising alternative for cisplatin-treated tumors [9]; though it exhibits reduced toxicity, this drug displays evidence of cross-resistance with cisplatin [7,10]. Common resistance mechanisms include increased nucleotide excision repair, loss of mismatch repair, and increased efflux of cisplatin, among others [7,8,11,12]. Interestingly, these resistance mechanisms can arise through cisplatin-induced mutations themselves, enabling cellular survival amidst cisplatin treatment [13–15]. Across various cell types and organisms, the mutation signature of cisplatin exhibits a high proportion of G:C to T:A transversion mutations, and the majority of cisplatin-induced mutations occur at typical sites of cisplatin-induced intrastrand cross-links (GpG, GpA, or ApG dinucleotides) [16–18]. These prominent G-to-T transversions can be explained in part by the misincorporation of dATP opposite the 5′-dG or 3′-dG of Pt-GG by error-prone DNA polymerases.

Notably, Y-family DNA polymerase η (polη) and human X-family DNA polymerase β (polβ) have been observed to contribute to cellular resistance to cisplatin treatment through both the accurate and mutagenic bypasses of Pt-GG intrastrand cross-links [19–24]. In agreement, polβ, which is a crucial component of base excision repair (BER), has been shown to exhibit significant increases in expression at both the transcriptomic and proteomic levels in multiple tumor types. In particular, polβ has been quantified up to protein levels of 286-fold in breast cancer tissues, among many other reports [25–30]. The overexpression of polβ has been thought to enhance its interference with lagging strand replication [31] and increase its activities in nucleotide excision repair [29] and error-prone translesion DNA synthesis [32]. Corroborating studies report that knockdown or inhibition of polβ sensitizes cells to cisplatin, underscoring the significant role polβ plays in modulating cellular sensitivity to cisplatin [33,34]. Importantly, polβ overexpression has also been demonstrated to increase genomic instability and result in a mutator phenotype in some cancer cells due to elevated polβ-mediated error-prone translesion synthesis that increases spontaneous mutations, emphasizing the role of polβ in broadening the landscape of intratumor genetic heterogeneity upon which cisplatin therapeutic selection can act, promoting the emergence of resistant clones composing the tumor population [32,35,36].

polβ has been previously demonstrated to cause significant misincorporation of dATP opposite the 5′-dG of gapped DNA containing an oxaliplatin-GG adduct, a similar but bulkier Pt-DNA adduct than Pt-GG [37]. Kinetic assays revealed this misincorporation is 17-fold higher in frequency across the major cisplatin-DNA adduct and showed that the Pt-GG adduct increases the frequency of dATP misinsertion relative to dTTP misinsertion, which is preferred on undamaged gapped DNA [37]. This further justifies the potential role of polβ as a key factor responsible for the mutagenic insertions that contribute to the prominent G-to-T transversions associated with the mutation signature of cisplatin. Therefore, the acquired resistance to cisplatin frequently observed at the cancer cell population-level may be, in part, due to the molecular-level consequences of the bipartite interplay between the cisplatin-induced formation of Pt-GG intrastrand cross-links and the mutagenic insertion opposite the 5′-G of Pt-GG by polβ. To advance understandings of this consequential mutagenic insertion by polβ across the major cisplatin-DNA adduct, particularly in polβ-overexpressing cancer cells, detailed structural characterizations are warranted.

We have previously reported crystal structures for the accurate nucleotide incorporation opposite Pt-GG, furthering understandings of polβ-mediated resistance to cisplatin [21]. However, the structural basis for the promutagenic catalysis across the major cisplatin-DNA adduct by polβ is not yet reported, despite the high yield of Pt-GG formation in cells during cisplatin treatment, the cisplatin-induced G-to-T mutation signature, and the significant dATP misinsertion previously observed across the 5′-dG. Herein, we report two crystal structures of polβ incorporating a non-hydrolyzable dATP analog opposite the 5′-dG of the templating Pt-GG cross-link, which reveal insights into the mutagenic bypass of cisplatin-DNA adducts by polβ.

Materials and methods

Preparation of the DNA duplex used for X-ray crystallographic studies

Oligonucleotides used for preparation of the modified DNA duplex were purchased from Integrated DNA Technologies (Coralville, IA). The sequence of the template DNA strand used for cisplatin modification and crystallographic studies was 5′-CCCACGGCCCATCACC-3′ (where GG designates the site of formation of the Pt-GG intrastrand cross-link by cisplatin). The previously reported method was used to prepare the platinum-modified template [38]. Briefly, a reaction containing a 2 : 1 molar ratio of silver nitrate to cisplatin in water was incubated 18 h in the dark at 37°C to produce active aquated cisplatin. The resulting silver chloride was removed and active cisplatin was isolated through centrifugation of the reaction mixture at 13 000 g for 10 min and collection of the supernatant. To modify the template DNA strand, 1.2-fold molar excess of the active cisplatin was mixed with the unmodified template DNA in 10 mM sodium phosphate (pH 6.8) and incubated in the dark for 18 h at 37°C. The reaction mixture was immediately subjected to ion exchange chromatography (Mono Q 5/50 GL, GE Healthcare), and the Pt-GG-modified template DNA was purified by elution over a gradient of 0.1 to 1.0 M NaCl in 10 mM Tris (pH 8.0). The purified Pt-GG-modified template DNA was desalted with a Sep-Pak C18 cartridge (Waters). The modified DNA was dried under reduced pressure at 30°C (Eppendorf, Vacufuge plus) and resuspended in water. The template strand containing the Pt-GG intrastrand cross-link was annealed with upstream and downstream primers in a 1 : 1 : 1 molar ratio to yield a single nucleotide gap, as described previously [39]. The upstream primer sequence was 5′-GGTGATGGGC-3′, and the downstream primer sequence was 5′-phosphate/GTGGG-3′.

Human DNA polymerase β expression and purification

Human polβ (amino acids 1–335) containing an N-terminal polyhistidine tag and Factor Xa cleavage site was expressed in E. coli BL21 (DE3) cells. Cultures were allowed to grow in LB at 37°C until OD600 reached 0.6. Cells were induced with 0.1 mM of isopropyl β-d-1-thiogalactopyranoside at 28°C for 18 h. Cells were pelleted at 4°C and lysed in HisTrap column binding buffer A (50 mM sodium phosphate (pH 7.8), 500 mM NaCl, 10% glycerol) containing 0.25 mM PMSF, 0.25% Nonidet-P-40, 0.25% Triton X-100, and 1 mg/ml lysozyme. Cell lysate was subjected to sonication for 60 s and centrifuged at 15 000 g at 4°C for 20 min. Protein was purified on a HisTrap HP column (GE Healthcare) and eluted in 500 mM imidazole. Protein was further purified on a Heparin HiTrap column (GE Healthcare) in 50 mM Tris ( pH 7.4), 10% glycerol, and 5 mM β-Mercaptoethanol and eluted in a gradient of 0.1–1 M NaCl. polβ was cleaved from its N-terminal tag by the addition of Factor Xa protease (NEB). Protein was subsequently purified by size exclusion chromatography (Superdex75, GE Healthcare), flash frozen in liquid nitrogen and stored at −80°C in 15 mg/ml aliquots in 20 mM Tris (pH 7.5), 10% glycerol, 200 mM NaCl, and 1 mM β-mercaptoethanol.

Crystallization of the polβ:Pt-GG•dATP ternary complexes

The hanging drop method was used for crystallography by mixing 1 μl of the polβ:Pt-GG complex solution with 1 μl of reservoir solution in the presence of 5 mM MgCl2 or MnCl2 and incoming non-hydrolyzable dATP* (dAMPNPP; Jena Bioscience). The crystals of the polβ:Pt-GG•dATP* complex were grown in 23% PEG 3350, 50 mM Tris (pH 6.5), and 0.35 M sodium acetate at 22°C for one week. Crystals were cryo-protected in reservoir buffer supplemented with 15% ethylene glycol and flash frozen in liquid nitrogen for data collection. Diffraction data sets for crystals of the ternary polβ:Pt-GG•dATP* complexes were collected at a wavelength of 0.97948 Å on beamline 5.0.3 at the Advanced Light Source (Berkeley, CA). Diffraction data for the polβ: Pt-GG•dATP* complexes was indexed and scaled using the program HKL2000. The data for each structure was further processed using CCP4 package programs. The structures were solved by molecular replacement using the ternary structure of the polβ:Pt-GG•dCTP* complex in the presence of Mg2+ (PDB ID: 4TUR) or Mn2+ (PDB ID: 4TUS) [21] as the search models. The location of the polβ:Pt-GG model with side chains added was optimized by several rounds of rigid body refinement. Manual model rebuilding was carried out with the Coot program into electron-density maps and refined using the CCP4 Refmac or PHENIX software suite. Crystallographic figures were prepared using Pymol. Statistics of data processing, data quality, and the refined models are summarized in Table 1.

Table 1.

Data collection and refinement statistics

PDB code Pt-GG•dATP-Mg2+ ternary 6U2O Pt-GG•dATP-Mn2+ ternary 6U6B
Space group P21 P21
Data collection Cell constants
  a (Å) 53.990 53.430
  b (Å) 79.184 78.216
  c (Å) 54.587 53.965
  α (°) 90.00 90.00
  β (°) 107.60 110.57
  γ (°) 90.00 90.00
 Resolution (Å)1 43.52–2.30 (2.34–2.30) 44.14–3.108 (3.15–3.10)
 Rmerge2 (%) 7.6 (34.6) 9.0 (45.0)
 <I/σ> 18.99 (2.61) 15.13 (2.58)
 Completeness (%) 100.0 (100.0) 100.0 (100.0)
 Redundancy 3.8 (3.8) 3.7 (3.8)
Refinement
 Rwork3/Rfree4(%) 20.10/24.81 22.22/26.75
 Unique reflections 17 892 7279
 Mean B factor (Å2)
  Protein 42.047 62.167
  Ligand 40.574 60.796
  Solvent 40.683 52.107
 Ramachandran plot
  Favored 94.72% 93.77%
  Additionally allowed 4.35% 5.92%
 R.M.S.D.
  Bond lengths (Å) 0.007 0.005
  Bond angles (°) 1.418 0.739
Open in a new tab
1

Values in parentheses represent the highest resolution shell.

2

Rmerge = Σ|I – |/ΣI, where I is the integrated intensity of a given reflection.

3

Rwork = Σ|F(obs) – F(calc)|/ΣF(obs).

4

Rfree = Σ|F(obs) – F(calc)|/ΣF(obs), calculated using 5% of the data.

Results and discussion

Ternary structure of polβ incorporating dATP opposite 5′-dG of the Pt-GG intrastrand cross-link in the presence of Mg2+

To understand the structural basis for the dATP misinsertion across the 5′-dG of the major cisplatin-DNA adduct by polβ [37] and to provide insights into the mutagenic bypass of this adduct, we solved a crystal structure of polβ inserting the non-hydrolyzable dATP analog dAMPNPP (dATP* hereafter) across the templating 5′-dG of the Pt-GG cross-link in the presence of Mg2+ (Figure 2). The polβ:Pt-GG•dATP*-Mg2+ ternary complex structure was refined to 2.3 Å resolution (Table 1). The Pt-GG-modified DNA duplex used here contained a single-nucleotide gap opposite the 5′G of the Pt-GG lesion and was of identical sequence as the DNA duplexes used previously in order to facilitate direct comparisons with reported polβ:Pt-GG•dCTP*, polβ: GG•dCTP*, and polβ : GG structures [21] (Figure 2A). The non-hydrolyzable dAMPNPP serves as an excellent isosteric substitute for dATP for crystallization and structural analyses. While usage of dATP would result in hydrolysis and subsequent nucleotidyl transfer, substitution with dATP* prevents catalysis through the presence of a bridging NH group. Importantly, this analog enables coordination of the primer terminus 3′-OH with the catalytic metal ion and otherwise does not affect the active site conformation of polβ, which is demonstrated in previous structural analyses of the pre-chemistry state of the enzyme’s active site [40–44].

Figure 2. Ternary structure of polβ incorporating dATP opposite the 5′G of the templating Pt-GG intrastrand cross-link with Mg2+.

Figure 2.

Open in a new tab

(A) DNA sequence used for crystallography. The downstream primer contains a 5′-phosphate that is not shown. The site representing nucleotide insertion opposite the 5′-G is indicated with an arrow. (B) Overall structure of the Pt-GG•dATP*-Mg2+ ternary complex (PDB ID: 6U2O). The protein is shown in white and its α-helix N is in red. The template strand is depicted in orange and the primer strands in yellow. The Pt-GG bases are shown in magenta, and the dC primer terminus is in green. The incoming dATP* is depicted in green. The Mg2+ and Na+ ions are shown in cyan and gray, respectively. (C) Active site of the Pt-GG•dATP*-Mg2+ ternary complex. The α-helix N is in a semi-open conformation. The Pt-GG adduct is depicted as spheres with Pt shown in gray and NH3 in blue. The distances between N2 of 5′-dG and N2 of 3′-dG, and between N2 of 5′-dG and the α-helix N are indicated. The angle between the N2 of 5′-dG and 3′-dG is indicated in blue. The published active site structures of (D) the unmodified GG gapped binary complex (PDB ID: 4TUP) [21]; (E) the unmodified GG•dCTP* complex (PDB ID: 4TUQ) [21]; (F) the Pt-GG•dCTP* complex (PDB ID: 4TUR) [21]. (G) Overlay of the various conformations of the α-helix N, including the closed unmodified GG•dCTP* complex (PDB ID: 4TUQ; light pink) [21], the semi-closed Pt-GG•dCTP*-Mn2+ complex (PDB ID: 4TUS; blue) [21], the semi-open Pt-GG•dATP*-Mg2+ complex (white), and the open unmodified GG gapped binary complex (PDB ID: 4TUP; smudge green) [21].

The Pt-GG•dATP*-Mg2+ complex adopts a ‘semi-open’ protein conformation that is similarly observed in the Pt-GG•dCTP*-Mg2+ complex (PDB ID: 4TUR) (Figure 2B–F) [21]. The semi-open protein conformation is characterized by a minor conformational change in the protein from the open conformation of the binary complex, specifically a 1.2–1.9 Å shift of the residues of the α-helix N towards the active site. Between the N2 of 3′-dG and N2 of 5′-dG, there is a large increase in distance to 9.5 Å (Figure 2C), as compared with previously reported structures. Additionally, there is a substantial roll angle of 85.1° between the 3′-dG and 5′-dG of the Pt-DNA adduct (Figure 2C), which is significantly larger than the 63.4° roll angle observed in the structure of the Pt-GG•dCTP* complex (Figure 2F). This is mostly due to an approximate 20° opening shift in the 5′-dG between the structures.

Strikingly, there is no canonical Watson–Crick base pairing exhibited between the incoming nucleotide and 5′-dG (Figure 3A,D). The incoming dATP appears to be in the mixture of the syn and anti conformations (Figure 3D). The incoming nucleotide does not form a coplanar base pairing with the templating 5′-G of Pt-GG. A water-mediated hydrogen bond between N7 of dATP* and Asn279 facilitates stabilization of the syn-dATP (Figure 3A). In addition, pi stacking interactions stabilize the binding of dATP* opposite the 5′-dG (Figure 3B). In particular, the incoming dATP* engages in edge-to-face interactions with the 5′-dG and primer terminus dC. There are also pi stacking interactions observed between the incoming nucleotide and Tyr271. Key residues are within hydrogen bonding distance of the phosphate backbone of dATP* for stabilization, including Arg183, Ser180, and Gly189 (Figure 3C). Within the active site, the distance between the 3′-OH of the primer terminus and the Pα of the incoming nucleotide is 3.3 Å, similar to the distance exhibited in the unmodified GG•dCTP* structure.

Figure 3. Active site structure of the Pt-GG•dATP*-Mg2+ ternary complex.

Figure 3.

Open in a new tab

(A) Close-up view of the active site of the Pt-GG•dATP*-Mg2+ ternary complex. Key residues are shown in white carbons. The nucleotide-binding ion is shown in cyan. Pt-GG is depicted in magenta carbons, and the primer terminus and incoming nucleotide are in green carbons. Water molecules are in red spheres. Hydrogen bonds are represented by dashed lines. (B) Close-up back-view of the active site and the stacking interactions governing the incorporation of the incoming nucleotide. Distances are indicated by blue arrows. (C) Close-up view of the interactions of the phosphate backbone of the incoming nucleotide with its environment. Metal ion coordination is shown in cyan. Key interactions are shown as dashed lines. Distance between the 3′-OH and Pα is indicated in blue. (D) Close-up view of the primer terminus base pair with the incoming nucleotide in both possible conformations (syn conformation in green, anti in yellow) and templating 5′-dG. The 2Fo – Fc electron density map is shown contoured at 1σ.

In the polβ:Pt-GG•dCTP*-Mg2+ complex structure, the catalytic metal ion (metal A) is absent and only the nucleotide-binding metal ion (metal B) is bound [45,46]. Due to the absence of the catalytic metal ion, there is a lack of coordination of both Asp256 and the 3′-OH with Mg2+, which has been shown to be a key interaction that facilitates proton transfer from the 3′-OH to Asp256 during catalysis [47]. Instead, the 3′-OH forms a hydrogen bond with O2 of Pα (2.6 Å) of the incoming nucleotide. Moreover, two water molecules are positioned in the vacant spot typically occupied by the absent metal ion, which assist in coordination of the 3′-OH, dATP*, and nearby residues. These observations suggest that catalytic incorporation of dATP by polβ in the presence of Mg2+ would be inefficient.

The overall structure of the Pt-GG•dATP*-Mg2+ complex is highly similar to the overall structure of the published Pt-GG•dCTP*-Mg2+ complex [21], with a root mean square deviation (r.m.s.d.) of 0.394 Å (Figure 4A and Table 2). Both structures reveal a semi-open protein conformation, and the key active site residues are similarly positioned (Figure 4B). Minor conformational differences are observed in the Lys280 and Arg283 residues, as well as the Pt-GG lesion. Lys280 is flipped towards the templating nucleotide and participates in its stabilization. Intriguingly, Arg283 adopts a different conformation for polβ and is positioned beneath the Pt-GG, rather than interacting with the O3′ of 5′-dG in Pt-GG•dCTP*-Mg2+. There are only minor differences in the DNA duplexes induced by the Pt-GG lesion between the correct and incorrect insertion structures, limited to the Pt-GG adduct and the dC 3′ to the lesion (Figure 4C), suggesting that misincorporation of dATP does not perturb the conformation of the DNA further than what is already altered by the major cisplatin-DNA adduct during the correct insertion.

Figure 4. Comparison of structures of polβ incorporating the correct (dCTP*) and incorrect (dATP*) nucleotides opposite the 5′G of the Pt-GG lesion in the presence of Mg2+.

Figure 4.

Open in a new tab

(A) Overlay of the overall structures of the Pt-GG•dATP*-Mg2+ ternary complex (multicolored) and the Pt-GG•dCTP*-Mg2+ ternary complex (light blue, PDB ID: 4TUR) [21]. (B) Overlay of the active site structures of the Pt-GG•dATP*-Mg2+ ternary complex (white carbons) and the Pt-GG•dCTP*-Mg2+ ternary complex (light blue). The Mg2+ ions are shown in white and light blue, respectively. Distances and angles are indicated in red. (C) Overlay of the gapped DNA duplexes of the Pt-GG•dATP*-Mg2+ ternary complex (yellow) and the Pt-GG•dCTP*-Mg2+ ternary complex (light blue).

Table 2.

Root mean square deviation values for structural comparisons of the dATP* misinsertion ternary complexes with previously reported structures

6U2O 6U6B
Reference PDB code Complex (conformation) r.m.s.d. (Å)
Koag et al. [21] 4TUP Undamaged GG binary complex (open) 0.285 0.982
Koag et al. [21] 4TUQ Undamaged GG•dCTP*-Mg2+ ternary complex (closed) 1.221 0.872
Koag et al. [21] 4TUR Pt-GG•dCTP*-Mg2+ ternary complex (semi-open) 0.394 1.359
Koag et al. [21] 4TUS Pt-GG•dCTP*-Mn2+ ternary complex (semi-closed) 1.143 0.844
Beard et al. [44] 3ISD THF•dATP*-Mn2+ ternary complex (closed) 1.250 0.763
Batra et al. [43] 3C2M dG•dATP*-Mn2+ ternary complex (closed) 1.202 0.845
– 6U2O Pt-GG•dATP*-Mg2+ ternary complex (semi-open) n/a 1.000
– 6U6B Pt-GG•dATP*-Mn2+ ternary complex (semi-closed) 1.000 n/a
Open in a new tab

In contrast, the Pt-GG•dATP*-Mg2+ complex varies considerably from the unmodified closed GG•dCTP* ternary complex (r.m.s.d. = 1.221 Å) (Figure 5A) [21]. As expected, the Pt-GG adduct significantly alters the conformation of the DNA duplex (Figure 5B). There are large variations in the base pairs upstream and downstream of the Pt-GG lesion. Moreover, the primer terminus is shifted further downstream by 1.6 Å. In the dATP* insertion structure, the minor groove recognition residues Asn279 and Arg283 do not participate in hydrogen-bonding interactions with the DNA (Figure 5C). Due to the semi-open conformation of the α-helix N, Asn279 is positioned further away from the incoming nucleotide and is not within hydrogen-bonding distance, but rather participates in the water-mediated hydrogen bonding interaction (Figure 3A). Unlike the structure of the unmodified GG ternary complex, Arg283 does not interact with the minor groove edge of the templating base. The Tyr271 residue of Pt-GG•dATP*-Mg2+ is positioned towards the imidazole ring of the incoming purine to participate in pi stacking interactions, rather than interacting with the primer terminus (Figure 5D).

Figure 5. Comparison of the structure of the Pt-GG•dATP*-Mg2+ ternary complex with the published structures of polβ in complex with unmodified DNA in the presence of Mg2+.

Figure 5.

Open in a new tab

(A) Overlay of the Pt-GG•dATP*-Mg2+ overall structure (multicolored) with the published unmodified GG•dCTP*-Mg2+ ternary complex overall structure (PDB ID: 4TUQ; light pink) [21]. (B) Overlay of the gapped DNA duplexes of the Pt-GG•dATP*-Mg2+ ternary complex (yellow) and the GG•dCTP*-Mg2+ ternary complex (light pink). Distances are indicated in red. (C) Overlay of the active site structures of the Pt-GG•dATP*-Mg2+ ternary complex (white carbons) and the unmodified GG•dCTP*-Mg2+ ternary complex (light pink). Distances and angles are indicated in red. The Mg2+ ions are shown in white and light pink, respectively. (D) Back-view of the active site structures (185° rotation of view in C). Tyr271 of Pt-GG•dATP* is indicated in yellow carbons and Tyr271 of the unmodified accurate ternary complex is in green carbons.

Overall, these molecular observations corroborate previous studies that highlight the sensitivity of polβ to bulky DNA adducts and its ability to adopt different conformations in response. Here, polβ recognizes the Pt-GG lesion and exhibits a semi-open conformation that appears to be sub-optimal for catalyzing the misin- sertion of dATP opposite the 5′-dG of Pt-GG.

Ternary structure of polβ incorporating dATP opposite 5′-dG of the Pt-GG intrastrand cross-link in the presence of Mn2+

The Pt-GG«dATP*-Mg2+ ternary complex structure most likely represents a ground state that must undergo a further metal-dependent open-to-closed protein conformational change for favorable catalysis [48]. In previously solved structures, we often observe the presence of one metal ion corresponds with an open protein conformation that is catalytically inefficient, whereas the binding of two metal ions may facilitate an open-to-closed conformational change to a catalytically competent state [21,46,49]. To gain further understandings of the polβ-mediated cisplatin-induced mutagenesis, we solved a crystal structure of polβ inserting dATP* across the templating 5′-dG of the Pt-GG adduct in the presence of the active site Mn2+. The Mn2+ ion has been demonstrated to promote the formation of a closed conformation during incorrect insertion [42,46,50]. The Pt-GG•dATP*-Mn2+ ternary complex structure was refined to 3.1 Å (Figure 6A).

Figure 6. Ternary structure of polβ incorporating dATP opposite the 5′G of the templating Pt-GG intrastrand cross-link with Mn2+.

Figure 6.

Open in a new tab

(A) Overall structure of the Pt-GG•dATP*-Mn2+ ternary complex (PDB ID: 6U6B). (B) Active site of the Pt-GG•dATP*-Mn2+ ternary complex. The α-helix N is in a semi-closed conformation. The distances between N2 of 5′-dG and N2 of 3′-dG, and between N2 of 5′-dG and the α-helix N are indicated. The angle between the N2 of 5′-dG and 3′-dG is indicated in blue. (C) Close-up view of the nascent base pair and minor groove-interacting residues. Distances are shown in red. Hydrogen bonds are represented by dashed lines. (D) Close-up view of the nascent base pair. The 2Fo – Fc electron density map is shown contoured at 1σ. Mn2+ ions are shown in cyan. (E) Close-up view of the active site of the Pt-GG•dATP*-Mn2+ ternary complex. The metal ions and coordination are shown in cyan. Distance between the 3′-OH and Pα is indicated by the blue arrow.

The Mn2+-bound ternary complex appears to be in a catalytically more favorable conformation than the Mg2+-bound ternary complex. The incoming dATP* is in a syn conformation and does not form a coplanar base pairing with the templating 5′G of Pt-GG (Figure 6B,C). As in the Mg2+-bound structure, the incoming dATP* engages in stacking interactions with Tyr271 and the primer terminus dC. Unlike the Mg2+-bound structure, both the catalytic and nucleotide-binding metal ions are present in the Mn2+-bound structure, which is evident in the electron density map (Figure 6B,D). The catalytic metal ion is co-ordinated with three catalytic carboxylates (Asp190, Asp192, Asp256), the primer terminus 3′-OH, and the phosphate oxygen. The α-helix N is in a semi-closed conformation rather than an open-conformation. The 3′-OH of primer terminus is 3.2 Å away from the Pα of dATP* and poised for in-line nucleophilic attack on the Pα (Figure 6E).

The Pt-GG•dATP*-Mn2+ ternary complex is significantly different from its Pt-GG•dATP*-Mg2+ counterpart (r.m.s.d. = 1.000 Å; Figure 7A), largely due to the change in protein conformation and position of the nascent base pair. In the active site, there is a slight variation in the position of the dATP*, as well as a corresponding shift in the nucleotide-binding ion (Figure 7B). There is a 2.1 Å upstream rise in the Pt atom, permitting a closer distance between the 3′- and 5′-dG of Pt-GG, which is also evident in the 3.8 Å rise in the phosphate and 2.8 Å rise in the O4′ of 5′-dG. Interestingly, this upstream shift of the Pt moiety and 5′-dG somewhat resembles the corresponding dG:dAMPCPP mismatch ternary structure (r.m.s.d. = 0.845 Å), where there is an upstream shift of the templating base from the coding template position, generating an abasic site intermediate (Figure 7C) [43]. However, the presence of the cis-diammineplatinum moiety appears to prevent the full upstream shift that would be typically achieved in its absence (Figure 7D). The primer terminus of the Pt-GG·dATP* complex does not rotate as significantly as that of the dG-dAMPCPP mismatch structure, enabling the formation of a much less staggered and more coplanar base pair with the 3′-dG. The combination of the constraints by the protein in the semi-closed conformation along with the bulkiness of the platinum moiety induce the compaction of the 3′-dG and 5′-dG of the Pt-GG intrastrand cross-link. Due to steric constraints of the intrastrand cross-link, this compaction is achieved through a tilting of the 5′-dG and an analogous adjustment in the dATP* to the syn conformation, which simultaneously supplies the aforementioned beneficial pi stacking interactions for the nucleotide’s stabilization. The tilting of the 5′-dG positions the base partially out of the templating base pocket and places it closer in distance to the 3′-dG. This in contrast with the 5′-dG of the undamaged mismatch structure, which fully vacates the coding base region and forms a single hydrogen bond with N6 of the incoming dATP*.

Figure 7. Comparison of the ternary structures of polβ misincorporating the dATP analog opposite the 5′G of the Pt-GG lesion.

Figure 7.

Open in a new tab

(A) Overlay of the overall structures of the Pt-GG•dATP*-Mn2+ (multicolored) and the Pt-GG•dATP*-Mg2+ ternary complex (teal). (B) Overlay of the active site structures of the Pt-GG•dATP*-Mn2+ ternary complex (white carbons) and the Pt-GG•dATP*-Mg2+ ternary complex (pale yellow). The Mn2+ and Mg2+ ions are shown in white and pale yellow, respectively. Distances and angles are indicated in red. (C) Overlay of the primer and nascent base pair structures of the Mn2+ ternary complex (white), the Mg2+ ternary complex (pale yellow), and the dG-dATP*-Mn2+ ternary complex (PDB ID: 3C2M; salmon). (D) Overlay of the active site structures of the Pt-GG•dATP*-Mn2+ ternary complex (white carbons) and the dG-dATP*-Mn2+ ternary complex (PDB ID: 3C2M; salmon). (E) Side-by-side comparison of B-factor analyses between the Mg2+ and Mn2+ active site structures.

Analyses of the crystallographic thermal B-factors between the Mg2+ and Mn2+ ternary structures reveal that the dATP* and 5′-dG are most labile in the Mg2+ complex, while the cisplatin moiety and 5′-dG are most labile in the Mn2+ complex (Figure 7E). The higher relative stability of the incoming dATP* in Pt-GG•dATP*-Mn2+ may be due to the presence of the catalytic metal ion, which can increase the stability of the incoming nucleotide. The position of the purine ring of the dATP* in the Mg2+ structure appears to be more flexible due to its lack of Watson–Crick base pairing with the 5′-dG, which also appears to be disordered. The primer terminus and one active-site metal of the Mg2+ structure are moderately well-ordered, each with an average B-factor of 39 and 35 Å2, respectively. In addition, the average B-factor of the Pt moiety is 34 Å2, indicating it is well-ordered, as compared with the Mn2+ structure. The average B-factors for the components of the Mn2+-bound active site structure are much higher, likely due to the low resolution of the structure. In addition, the semi-closed conformation of the α-helix N in the Mn2+ structure may contribute to the high B-factor of the Pt atom through the constraints by the protein and resultant compaction of the 5′-dG towards the 3′-dG, as demonstrated by the shorter distance between the 3′-dG and 5′-dG than that of other Pt-GG structures (Figure 2C,F and 6B).

Overall, the observation of the catalytic metal coordination, the semi-closed conformation, and syn-dATP in the Mn2+-bound structure suggest that the insertion of dATP opposite the Pt-GG adduct may occur without involving a coplanar base pairing.

The major cisplatin lesion behaves like an abasic site in polβ active site

Our data presented here reveal insights into cisplatin-induced mutagenesis by human DNA polymerase β. Our structures depict — within the confines of the polβ active site — how the Pt-GG intrastrand cross-link promotes misinsertion of dATP across the 5′-dG. In contrast with canonical Watson–Crick base pairing characteristics, the structures we present here are characterized by non-instructional interactions such as protein templating and pi stacking interactions with the DNA, which are similarly observed in the ternary structure of polβ-mediated misinsertion of dAMPCPP opposite an abasic site analog, tetrahydrofuran (THF) [44]. Specifically, in the catalytically competent Mn2+ structure, we observe an altered conformation of the Arg283 side chain. While Arg283 typically interacts with the N3 of the templating 5′-dG and O4′ of the 3′-dG in the closed GG•dCTP* ternary structure of polβ, we observe the side chain of Arg283 only forms interactions with O3′ of 5′-dG and not with the nucleobase. Instead, Lys280 is positioned well to weakly interact with the N3 of 5′-dG within both the Mg2+ and Mn2+ structures (Figure 7B). Typically, Lys280 stabilizes templating purines through van der Waals interactions [51]. Our observations indicate Lys280 plays a previously unobserved role in potentially forming a hydrogen bond to stabilize the templating 5′-dG. These weak interactions of the protein with the 5′-dG may allow for greater flexibility of the templating base. Furthermore, several pi stacking interactions occur and stabilize the purine moiety of the incoming nucleotide (Figure 3B and 6C). The incoming dATP* forms edge-to-face interactions with the templating 5′-dG, primer terminus dC and Tyr271. These pi stacking interactions and hydrogen bonding interactions of dATP* with Tyr271 are similarly observed in the structure of polβ-mediated misinsertion of dAMPCPP opposite an abasic site analog [44]. Together, these protein templating and pi stacking interactions drive the inefficient mutagenic insertion of dATP.

The structure of the Pt-GG•dATP*-Mn2+ ternary complex superimposes most similarly to the structure of the abasic site insertion (r.m.s.d. = 0.763 Å) (Figure 8A and Table 2). In both structures, the protein adopts a semi-closed conformation. The template strands both experience a shift upstream compared with the ternary structure of polβ correctly inserting dCTP across an unmodified templating dG. The bases of the Pt-GG adduct shift upstream ~2.5 Å in the presence of the dATP analog compared with the position of undamaged DNA upon binding the correct dCTP (not shown), albeit the template strand of the THF-dATP* complex is shifted upstream to a greater degree. Most notably, Tyr271 of both structures is pointed away from the primer terminus to form dual interactions with the incoming dATP analog (Figure 8B). The hydroxyl group of Tyr271 is within weak hydrogen bonding distance to the N7 or N3 of dATP* of the Pt-GG or abasic site structure, respectively; the tyrosyl ring is poised to form pi stacking interactions with the purine moiety in both structures, assisting in the nucleotide’s stabilization where base pairing is absent. While the incoming dATP* of the THF-dATP* complex is in the anti conformation, the dATP* bound across from the Pt-GG adduct benefits from a stabilizing edge-to-face interaction with the primer terminus when it adopts the syn conformation (Figure 8A). This also in tandem allows for a weak hydrogen bond of N1 of dATP* with N2 of the 3′-dG for further stability (Figure 8A).

Figure 8. Comparison of the Pt-GG•dATP*-Mn2+ structure with published structure of polβ-mediated misinsertion of dATP opposite THF, an abasic site analog.

Figure 8.

Open in a new tab

(A) Overlay of the active site structures of the Pt-GG•dATP*-Mn2+ ternary complex (white) and THF-dATP*-Mn2+ ternary complex (PDB ID: 3ISD; red) [44]. The abasic site analog is shown in blue. Distances are shown in blue. Nearby residues are depicted in cartoon loop and sticks. (B) Comparison of the position of Tyr271 residues between the two structures. The Tyr271 of THF-dATP*-Mn2+ is depicted in green carbons and Tyr271 of Pt-GG•dATP*-Mn2+ is depicted in white carbons. Hydrogen bonds are represented by dashed lines.

In the case of dATP misincorporation opposite an abasic site by polβ, this ternary structure offers understandings of a protein templating basis, where the polymerase contributes hydrogen bonding interactions and geometric constraints to stabilize and favor insertion of the incoming dATP. Here, the structures of polβ misincorporating dATP opposite the Pt-GG intrastrand cross-link also demonstrate that protein templating, particularly an interaction with Lys280, in addition to pi stacking interactions of the purine with nearby bases and Tyr271, favor the mutagenic insertion. In addition, as a purine, adenine is better suited to form pi stacking interactions with its environment than a pyrimidine, which cannot contribute as significantly to stacking and hydrophobic interactions. In support of this notion, a previous report shows adenine exhibits a lower dipole moment than other bases, enabling it to contribute markedly to stacking interactions [52]. Overall, the empty space within the nascent base pair binding pocket is best filled with adenine due to its steric bulkiness as a purine and abilities to form pi stacking interactions. With a role for polβ in mutagenic replication past other DNA lesions [49,53,54], once again we are poised with evidence to appreciate the sensitivity of polβ in binding inaccurate nucleotides across bulky DNA lesions with minor alterations in protein conformation.

Conclusion

Here, we report the first structures of a DNA polymerase bypassing the major cisplatin-DNA lesion, specifically polβ-catalyzed misincorporation of dATP opposite the 5′-dG of the Pt-GG intrastrand cross-link. In the polβ active site, dATP does not form a coplanar base pairing with the 5′-dG of Pt-GG. The incoming dATP opposite the 5′-dG is accommodated via pi stacking interactions and protein templating. We also note similarities between the dATP misinsertion opposite Pt-GG lesion and the dATP misinsertion opposite an abasic site, largely due to the upstream shift in the template strand and the role of Tyr271 in stabilizing the incoming purine. Our results, together with the reported overexpression of polβ in tumors, provide insights into cisplatin-induced mutagenesis, particularly in cisplatin-treated polβ-overexpressing tumors, where this misinsertion may be inefficient yet more pervasive.

Acknowledgements

We thank Myong-Chul Koag, who made the polβ construct and assisted with the polβ-DNA crystallization. We thank Dr. Arthur Monzingo for technical assistance and the Macromolecular Crystallography Facility for instrumentation and technical assistance, with financial support from the College of Natural Sciences, the Office of the Executive Vice President and Provost and the Institute for Cellular and Molecular Biology at the University of Texas at Austin. The Berkeley Center for Structural Biology is supported in part by the National Institute of General Medical Sciences of the National Institute of Health. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Funding

This work was supported by the National Institutes of Health [ES26676]. Funding for open access charge: [ES26676]. C.K.V. is supported by the University of Texas at Austin Provost’s Graduate Excellence Fellowship and the American Association of University Women Austin Branch Doctoral Fellowship, and the F31 Ruth L. Kirschstein NRSA Predoctoral Fellowship through NCI.

Abbreviations

BER

base excision repair

Pt-DNA

platinum-DNA

THF

tetrahydrofuran

Footnotes

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Data Availability

PDB IDs: 6U2O and 6U6B.

References

  • 1.Rabik CA and Dolan ME (2007) Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat. Rev. 33, 9–23 10.1016/j.ctrv.2006.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Wheate NJ, Walker S, Craig GE and Oun R (2010) The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 39, 8113 10.1039/c0dt00292e [DOI] [PubMed] [Google Scholar]
  • 3.Dasari S and Tchounwou PB (2014) Cisplatin in cancer therapy: molecular mechanisms of action. Eur. J. Pharmacol. 740, 364–378 10.1016/j.ejphar.2014.07.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wang D and Lippard SJ (2005) Cellular processing of platinum anticancer drugs. Nat. Rev. Drug Discov. 4, 307–320 10.1038/nrd1691 [DOI] [PubMed] [Google Scholar]
  • 5.Jung Y and Lippard SJ (2007) Direct cellular responses to platinum-induced DNA damage. Chem. Rev. 107, 1387–1407 10.1021/cr068207j [DOI] [PubMed] [Google Scholar]
  • 6.Rocha CRR, Silva MM, Quinet A, Cabral-Neto JB and Menck CFM (2018) DNA repair pathways and cisplatin resistance: an intimate relationship. Clinics (Sao Paulo) 73, e478s 10.6061/clinics/2018/e478s [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O et al. (2012) Molecular mechanisms of cisplatin resistance. Oncogene 31, 1869–1883 10.1038/onc.2011.384 [DOI] [PubMed] [Google Scholar]
  • 8.Christie EL and Bowtell DDL (2017) Acquired chemotherapy resistance in ovarian cancer. Ann. Oncol. 28, viii13–viii15 10.1093/annonc/mdx446 [DOI] [PubMed] [Google Scholar]
  • 9.Oun R, Moussa YE and Wheate NJ (2018) The side effects of platinum-based chemotherapy drugs: a review for chemists. Dalton Trans. 47, 6645–6653 10.1039/C8DT00838H [DOI] [PubMed] [Google Scholar]
  • 10.Gore M, Fryatt I, Wiltshaw E, Dawson T, Robinson B and Calvert A (1989) Cisplatin/carboplatin cross-resistance in ovarian cancer. Br. J. Cancer 60, 767–769 10.1038/bjc.1989.356 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kelland L (2007) The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 7, 573–584 10.1038/nrc2167 [DOI] [PubMed] [Google Scholar]
  • 12.Shen D-W, Pouliot LM, Hall MD and Gottesman MM (2012) Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol. Rev. 64, 706–721 10.1124/pr.111.005637 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Sakai W, Swisher EM, Karlan BY, Agarwal MK, Higgins J, Friedman C, et al. (2008) Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers. Nature 451, 1116–1120 10.1038/nature06633 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Swisher EM, Sakai W, Karlan BY, Wurz K, Urban N and Taniguchi T (2008) Secondary BRCA1 mutations in BRCA1-mutated ovarian carcinomas with platinum resistance. Cancer Res. 68, 2581–2586 10.1158/0008-5472.CAN-08-0088 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Xie K, Doles J, Hemann MT and Walker GC (2010) Error-prone translesion synthesis mediates acquired chemoresistance. Proc. Natl Acad. Sci. U.S.A. 107, 20792–20797 10.1073/pnas.1011412107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014) C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24, 1624–1636 10.1101/gr.175547.114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Szikriszt B, Póti Á, Pipek O, Krzystanek M, Kanu N, Molnár J, et al. (2016) A comprehensive survey of the mutagenic impact of common cancer cytotoxics. Genome Biol. 17, 99 10.1186/s13059-016-0963-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Boot A, Huang MN, Ng AWT, Ho S-C, Lim JQ, Kawakami Y et al. (2018) In-depth characterization of the cisplatin mutational signature in human cell lines and in esophageal and liver tumors. Genome Res. 28, 654–665 10.1101/gr.230219.117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zhao Y, Biertumpfel C, Gregory MT, Hua Y-J, Hanaoka F and Yang W (2012) Structural basis of human DNA polymerase -mediated chemoresistance to cisplatin. Proc. Natl Acad. Sci. U.S.A. 109, 7269–7274 10.1073/pnas.1202681109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Ummat A, Rechkoblit O, Jain R, Roy Choudhury J, Johnson RE, Silverstein TD, et al. (2012) Structural basis for cisplatin DNA damage tolerance by human polymerase η during cancer chemotherapy. Nat. Struct. Mol. Biol. 19, 628–632 10.1038/nsmb.2295 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Koag M-C, Lai L and Lee S (2014) Structural basis for the inefficient nucleotide incorporation opposite cisplatin-DNA lesion by human DNA polymerase β. J. Biol. Chem. 289, 31341–31348 10.1074/jbc.M114.605451 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hoffmann JS, Pillaire MJ, Garcia-Estefania D, Lapalu S and Villani G (1996) In vitro bypass replication of the cisplatin-d(GpG) lesion by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase is highly mutagenic. J. Biol. Chem. 271, 15386–15392 10.1074/jbc.271.26.15386 [DOI] [PubMed] [Google Scholar]
  • 23.Vaisman A, Lim SE, Patrick SM, Copeland WC, Hinkle DC, Turchi JJ et al. (1999) Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts. Biochemistry 38, 11026–11039 10.1021/bi9909187 [DOI] [PubMed] [Google Scholar]
  • 24.Vaisman A, Masutani C, Hanaoka F and Chaney SG (2000) Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase η. Biochemistry 39, 4575–4580 10.1021/bi000130k [DOI] [PubMed] [Google Scholar]
  • 25.Srivastava DK (1999) DNA polymerase expression differences in selected human tumors and cell lines. Carcinogenesis 20, 1049–1054 10.1093/carcin/20.6.1049 [DOI] [PubMed] [Google Scholar]
  • 26.Bergoglio V, Canitrot Y, Hogarth L, Minto L, Howell SB, Cazaux C et al. (2001) Enhanced expression and activity of DNA polymerase β in human ovarian tumor cells: impact on sensitivity towards antitumor agents. Oncogene 20, 6181–6187 10.1038/sj.onc.1204743 [DOI] [PubMed] [Google Scholar]
  • 27.Kraker AJ and Moore CW (1988) Elevated DNA polymerase beta activity in a cis-diamminedichloroplatinum(II) resistant P388 murine leukemia cell line. Cancer Lett. 38, 307–314 10.1016/0304-3835(88)90022-5 [DOI] [PubMed] [Google Scholar]
  • 28.Canitrot Y, Lautier D, Laurent G, Fréchet M, Ahmed A, Turhan A et al. (1999) Mutator phenotype of BCR – ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase β. Oncogene 18, 2676–2680 10.1038/sj.onc.1202619 [DOI] [PubMed] [Google Scholar]
  • 29.Canitrot Y, Hoffmann JS, Calsou P, Hayakawa H, Salles B and Cazaux C (2000) Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells. FASEB J. 14, 1765–1774 10.1096/fj.99-1063com [DOI] [PubMed] [Google Scholar]
  • 30.Servant L, Cazaux C, Bieth A, Iwai S, Hanaoka F and Hoffmann J-S (2002) A role for DNA polymerase β in mutagenic UV lesion bypass. J. Biol. Chem. 277, 50046–50053 10.1074/jbc.M207101200 [DOI] [PubMed] [Google Scholar]
  • 31.Servant L, Bieth A, Hayakawa H, Cazaux C and Hoffmann J-S (2002) Involvement of DNA polymerase β in DNA replication and mutagenic consequences. J. Mol. Biol. 315, 1039–1047 10.1006/jmbi.2001.5307 [DOI] [PubMed] [Google Scholar]
  • 32.Canitrot Y, Cazaux C, Frechet M, Bouayadi K, Lesca C, Salles B et al. (1998) Overexpression of DNA polymerase in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs. Proc. Natl Acad. Sci. U.S.A. 95, 12586–12590 10.1073/pnas.95.21.12586 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Horton JK, Srivastava DK, Zmudzka BZ and Wilson SH (1995) Strategic down-regulation of DNA polymerase β by antisense RNA sensitizes mammalian cells to specific DNA damaging agents. Nucleic Acids Res. 23, 3810–3815 10.1093/nar/23.19.3810 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Boudsocq F (2005) Modulation of cellular response to cisplatin by a novel inhibitor of DNA polymerase. Mol. Pharmacol. 67, 1485–1492 10.1124/mol.104.001776 [DOI] [PubMed] [Google Scholar]
  • 35.Canitrot Y, Fréchet M, Servant L, Cazaux C and Hoffmann J-S (1999) Overexpression of DNA polymerase β: a genomic instability enhancer process. FASEB J. 13, 1107–1111 10.1096/fasebj.13.9.1107 [DOI] [PubMed] [Google Scholar]
  • 36.Chan K, Houlbrook S, Zhang Q-M, Harrison M, Hickson ID and Dianov GL (2007) Overexpression of DNA polymerase results in an increased rate of frameshift mutations during base excision repair. Mutagenesis 22, 183–188 10.1093/mutage/gel070 [DOI] [PubMed] [Google Scholar]
  • 37.Vaisman A, Warren MW and Chaney SG (2001) The effect of DNA structure on the catalytic efficiency and fidelity of human DNA polymerase β on templates with platinum-DNA adducts. J. Biol. Chem. 276, 18999–19005 10.1074/jbc.M007805200 [DOI] [PubMed] [Google Scholar]
  • 38.Wang D, Hara R, Singh G, Sancar A and Lippard SJ (2003) Nucleotide excision repair from site-specifically platinum-modified nucleosomes. Biochemistry 42, 6747–6753 10.1021/bi034264k [DOI] [PubMed] [Google Scholar]
  • 39.Sawaya MR, Prasad R, Wilson SH, Kraut J and Pelletier H (1997) Crystal structures of human DNA polymerase β complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36, 11205–11215 10.1021/bi9703812 [DOI] [PubMed] [Google Scholar]
  • 40.Batra VK, Beard WA, Shock DD, Krahn JM, Pedersen LC and Wilson SH (2006) Magnesium-induced assembly of a complete DNA polymerase catalytic complex. Structure 14, 757–766 10.1016/j.str.2006.01.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chamberlain BT, Batra VK, Beard WA, Kadina AP, Shock DD, Kashemirov BA et al. (2012) Stereospecific formation of a ternary complex of (S)-α,β-fluoromethylene-dATP with DNA pol β. Chembiochem 13, 528–530 10.1002/cbic.201100738 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Freudenthal BD, Beard WA, Shock DD and Wilson SH (2013) Observing a DNA polymerase choose right from wrong. Cell 154, 157–168 10.1016/j.cell.2013.05.048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Batra VK, Beard WA, Shock DD, Pedersen LC and Wilson SH (2008) Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation. Mol. Cell 30, 315–324 10.1016/j.molcel.2008.02.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Beard WA, Shock DD, Batra VK, Pedersen LC and Wilson SH (2009) DNA polymerase beta substrate specificity: side chain modulation of the “A-rule.”. J. Biol. Chem. 284, 31680–31689 10.1074/jbc.M109.029843 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Freudenthal BD, Beard WA and Wilson SH (2012) Structures of dNTP intermediate states during DNA polymerase active site assembly. Structure 20, 1829–1837 10.1016/j.str.2012.08.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Koag M-C and Lee S (2014) Metal-dependent conformational activation explains highly promutagenic replication across O6-methylguanine by human DNA polymerase β. J. Am. Chem. Soc. 136, 5709–5721 10.1021/ja500172d [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Batra VK, Perera L, Lin P, Shock DD, Beard WA, Pedersen LC et al. (2013) Amino acid substitution in the active site of DNA polymerase β explains the energy barrier of the nucleotidyl transfer reaction. J. Am. Chem. Soc. 135, 8078–8088 10.1021/ja403842j [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Lin P, Batra VK, Pedersen LC, Beard WA, Wilson SH and Pedersen LG (2008) Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase. Proc. Natl Acad. Sci. U.S.A. 105, 5670–5674 10.1073/pnas.0801257105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Koag M-C, Kou Y, Ouzon-Shubeita H and Lee S (2014) Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine. Nucleic Acids Res. 42, 8755–8766 10.1093/nar/gku554 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Vaisman A, Ling H, Woodgate R and Yang W (2005) Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis. EMBO J. 24, 2957–2967 10.1038/sj.emboj.7600786 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Beard WA, Shock DD, Yang X-P, DeLauder SF and Wilson SH (2002) Loss of DNA polymerase β stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity. J. Biol. Chem. 277, 8235–8242 10.1074/jbc.M107286200 [DOI] [PubMed] [Google Scholar]
  • 52.Guckian KM, Schweitzer BA, Ren RX-F, Sheils CJ, Tahmassebi DC and Kool ET (2000) Factors contributing to aromatic stacking in water: evaluation in the context of DNA. J. Am. Chem. Soc. 122, 2213–2222 10.1021/ja9934854 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Batra VK, Shock DD, Beard WA, McKenna CE and Wilson SH (2012) Binary complex crystal structure of DNA polymerase reveals multiple conformations of the templating 8-oxoguanine lesion. Proc. Natl Acad. Sci. U.S.A. 109, 113–118 10.1073/pnas.1112235108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Koag M-C, Jung H and Lee S (2019) Mutagenic replication of the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine by human DNA polymerases. J. Am. Chem. Soc. 141, 4584–4596 10.1021/jacs.8b08551 [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES