Kyle L. Brown

Chemistry and Structural Biology of DNA Damage and Biological Consequences

The formation of adducts by the reaction of chemicals with DNA is a critical step for the initiation of carcinogenesis. The structural analysis of various DNA adducts reveals that conformational and chemical rearrangements and interconversions are a common theme. Conformational changes are modulated both by the nature of adduct and the base sequences neighboring the lesion sites. Equilibria between conformational states may modulate both DNA repair and error‐prone replication past these adducts. Likewise, chemical rearrangements of initially formed DNA adducts are also modulated both by the nature of adducts and the base sequences neighboring the lesion sites. In this review, we focus on DNA damage caused by a number of environmental and endogenous agents, and biological consequences.

Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5′-GTgG-3′ sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson–Crick alignment. The Tg N3H imine and A N6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3′-neighbor G•C base pair is disrupted. The solvent accessible surface and T2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3′-neighbor guanine alleviate steric clash with the 5′-neighbor base pair. Tg also destabilizes the 5′-neighbor G•C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

Interconversion of the cis-5R,6S-and trans-5R,6R.Thymine Glycol Lesions in Duplex DNA

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5R Tg lesion was incorporated site-specifically into 5′-d(G1T2G3C4G5Tg6G7T8T9T10G11T12)-3′; Tg = 5R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5′-d(A13C14A15A16A17C18A19C20G21C22A23C24)-3′, forming the Tg6•A19 base pair, corresponding to the oxidative damage of thymine in DNA, or 5′-d(A13C14A15A16A17C18G19C20G21C22A23C24)-3′, forming the mismatched Tg6•G19 base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA. At 30 °C, the equilibrium ratio of cis-5R,6S:trans-5R,6R epimers was 7:3 for the duplex containing the Tg6•A19 base pair. In contrast, for the duplex containing the Tg6•G19 base pair, the cis-5R,6S:trans-5R,6R equilibrium favored the cis-5R,6S epimer; the level of the trans-5R,6R epimer remained below the level of detection by NMR. The data suggested that Tg disrupted hydrogen bonding interactions, either when placed opposite to A19 or G19. Thermodynamic measurements indicated a 13 °C reduction of Tm regardless of whether Tg was placed opposite dG or dA in the complementary strand. Although both pairings increased the free energy of melting by 3 kcal/mol, the melting of the Tg•G pair was more enthalpically favored than was the melting of the Tg•A pair. The observation that the position of the equilibrium between the cis-5R,6S and trans-5R,6R thymine glycol epimers in duplex DNA was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of Tg [Ocampo-HafallaM. T.; AltamiranoA.; BasuA. K.; ChanM. K.; OcampoJ. E.; CummingsA.Jr.; BoorsteinR. J.; CunninghamR. P.; TeeborG. W.DNA Repair (Amst)2006, 5, 444−454].

Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, we conclude that collagen IV and its variant, spongin, are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis. DOI: http://dx.doi.org/10.7554/eLife.24176.001

Hypohalous Acids Contribute to Renal Extracellular Matrix Damage in Experimental Diabetes

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid–derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1β1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W28 and W192 are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid–derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.

Extracellular chloride signals collagen IV network assembly during basement membrane formation

Chloride is ubiquitous in physiology but understood to provide ionic strength for tissue function. The authors discover a molecular function of chloride whereby the ion signals the assembly of collagen IV, establishing a microenvironment on the outside of cells.

Bypass of aflatoxin B1 adducts by the Sulfolobus solfataricus DNA polymerase IV.

Aflatoxin B1 (AFB1) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB1–N7-dG). The AFB1–N7-dG can rearrange to a formamidopyrimidine (AFB1–FAPY) derivative. Both AFB1–N7-dG and the β-anomer of the AFB1–FAPY adduct yield G→T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB1–N7-dG in an error-free manner but conducts error-prone replication past the AFB1–FAPY adduct, including misinsertion of dATP, consistent with the G→T mutations observed in E. coli. Three ternary (Dpo4–DNA–dNTP) structures with AFB1–N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB1–N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5′-template neighbor dT from a primed AFB1–N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O2 oxygen of dTTP, and between the template T O4 oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB1–N7-dG maintains the 5′-intercalation of the AFB1 moiety observed in DNA. The bond between N7-dG and C8 of the AFB1 moiety remains in plane with the alkylated guanine, creating a 16° inclination of the AFB1 moiety with respect to the guanine. A binary (Dpo4–DNA) structure with an AFB1–FAPY adducted template:primer also maintains 5′-intercalation of the AFB1 moiety. The β-deoxyribose anomer is observed. Rotation about the FAPY C5–N5 bond orients the bond between N5 and C8 of the AFB1 moiety out of plane in the 5′-direction, with respect to the FAPY base. The formamide group extends in the 3′-direction. This improves stacking of the AFB1 moiety above the 5′-face of the FAPY base, as compared to the AFB1–N7-dG adduct. Ternary structures with AFB1–β-FAPY adducted template:primers show correct vs incorrect insertion of dATP vs dTTP opposite the 5′-template neighbor dT from a primed AFB1–β-FAPY:dC pair. For dATP, the oxygen atom of the FAPY formamide group participates in a water-mediated hydrogen bond with Arg332. The insertion of dTTP yields a structure similar to that observed for the AFB1–N7-dG adduct. The differential accommodation of these AFB1 adducts within the active site may, in part, modulate lesion bypass.

The cis-(5R,6S)-Thymine Glycol Lesion Occupies the Wobble Position When Mismatched with Deoxyguanosine in DNA

Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, mispaired with deoxyguanosine. The structure of the 5R Tg·G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5′-d(G1T2G3C4G5Tg6G7T8T9T10G11T12)-3′·5′-d(A13C14A15A16A17C18G19C20G21C22A23C24)-3′; Tg = 5R Tg. In this duplex the cis-5R,6S:trans-5R,6R equilibrium favors the cis-5R,6S epimer [Brown, K. L., Adams, T., Jasti, V. P., Basu, A. K., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 11701−11710]. The cis-5R,6S Tg lesion is in the wobble orientation such that Tg6O2 is proximate to G19 N1H and Tg6 N3H is proximate to G19O6. Both Tg6 and the mismatched nucleotide G19 remain stacked in the helix. The Tg6 nucleotide shifts toward the major groove and stacks below the 5′-neighbor base G5, while its complement G19 stacks below the 5′-neighbor C20. In the 3′-direction, stacking between Tg6 and the G7·C18 base pair is disrupted. The solvent-accessible surface area of the Tg nucleotide increases as compared to the native Watson−Crick hydrogen-bonded T·A base pair. An increase in T2 relaxation rates for the Tg6 base protons is attributed to puckering of the Tg base, accompanied by increased disorder at the Tg·G mismatch pair. The axial vs equatorial conformation of the Tg6 CH3 group cannot be determined with certainty from the NMR data. The rMD trajectories suggest that in either the axial or equatorial conformations the cis-5R,6S Tg lesion does not form strong intrastrand hydrogen bonds with the imidazole N7 atom of the 3′-neighbor purine G7. The wobble pairing and disorder of the Tg·G mismatch correlate with the reduced thermodynamic stability of the mismatch and likely modulate its recognition by DNA base excision repair systems.

Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

Aflatoxin B1 (AFB1) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB1–N7-dG). The AFB1–N7-dG can rearrange to a formamidopyrimidine (AFB1–FAPY) derivative. Both AFB1–N7-dG and the β-anomer of the AFB1–FAPY adduct yield G→T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB1–N7-dG in an error-free manner but conducts error-prone replication past the AFB1–FAPY adduct, including misinsertion of dATP, consistent with the G→T mutations observed in E. coli. Three ternary (Dpo4–DNA–dNTP) structures with AFB1–N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB1–N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5′-template neighbor dT from a primed AFB1–N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O2 oxygen of dTTP, and between the template T O4 oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB1–N7-dG maintains the 5′-intercalation of the AFB1 moiety observed in DNA. The bond between N7-dG and C8 of the AFB1 moiety remains in plane with the alkylated guanine, creating a 16° inclination of the AFB1 moiety with respect to the guanine. A binary (Dpo4–DNA) structure with an AFB1–FAPY adducted template:primer also maintains 5′-intercalation of the AFB1 moiety. The β-deoxyribose anomer is observed. Rotation about the FAPY C5–N5 bond orients the bond between N5 and C8 of the AFB1 moiety out of plane in the 5′-direction, with respect to the FAPY base. The formamide group extends in the 3′-direction. This improves stacking of the AFB1 moiety above the 5′-face of the FAPY base, as compared to the AFB1–N7-dG adduct. Ternary structures with AFB1–β-FAPY adducted template:primers show correct vs incorrect insertion of dATP vs dTTP opposite the 5′-template neighbor dT from a primed AFB1–β-FAPY:dC pair. For dATP, the oxygen atom of the FAPY formamide group participates in a water-mediated hydrogen bond with Arg332. The insertion of dTTP yields a structure similar to that observed for the AFB1–N7-dG adduct. The differential accommodation of these AFB1 adducts within the active site may, in part, modulate lesion bypass.

Structural Perturbations Induced by the α-Anomer of the Aflatoxin B1 Formamidopyrimidine Adduct in Duplex and Single-Strand DNA

The guanine N7 adduct of aflatoxin B1exo-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between α and β anomers. The β anomer is highly mutagenic in Escherichia coli, producing G → T transversions; it thermally stabilizes the DNA duplex. The AFB-α-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-α-FAPY adduct has been elucidated in 5′-d(C1T2A3T4X5A6T7T8C9A10)-3′·5′-d(T11G12A13A14T15C16A17T18A19G20)-3′ (X = AFB-α-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5′ face of the pyrimidine moiety at the damaged nucleotide between base pairs T4·A17 and X5·C16, placing the FAPY C5−N5 bond in the Ra axial conformation. Large perturbations of the ε and ζ backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5′ neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3′-neighbor adenine stabilizes the E conformation of the formamide moiety. NMR studies reveal a similar 5′-intercalation of the AFB moiety for the AFB-α-FAPY adduct in the tetramer 5′-d(C1T2X3A4)-3′, involving the Ra axial conformation of the FAPY C5−N5 bond and the E conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-β-FAPY adduct also intercalates on the 5′ side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5′-stacking leads to the Ra conformational preference about the C5−N5 bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-α-FAPY adduct explain its lower stability as compared to the AFB-β-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3′-neighboring adenine stabilizing the E configuration of the formamide moiety is also observed for the AFB-β-FAPY adduct, and suggests that the identity of the 3′-neighbor nucleotide modulates the stability and biological processing of AFB adducts.