Karen C. Angel

DNA Adduct Bypass Polymerization by Sulfolobus Solfataricus DNA Polymerase Dpo4: Analysis and Crystal Structures of Multiple Base Pair Substitution and Frameshift Products with the Adduct 1,N2-Ethenoguanine.

1,N2-Etheno(ϵ)guanine is a mutagenic DNA lesion derived from lipid oxidation products and also from some chemical carcinogens. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3′-(1,N2-ϵ-G)TACT-5′, among the four dNTPs tested individually. With the template 3′-(1,N2-ϵ-G)CACT-5′, both G and A were incorporated. When primer extension was done in the presence of a mixture of all four dNTPs, high pressure liquid chromatography-mass spectrometry analysis of the products indicated that (opposite 3′-(1,N2-ϵ-G)CACT-5′) the major product was 5′-GTGA-3′ and the minor product was 5′-AGTGA-3′. With the template 3′-(1,N2-ϵ-G)TACT-5′, the following four products were identified by high pressure liquid chromatography-mass spectrometry: 5′-AATGA-3′, 5′-ATTGA-3′, 5′-ATGA-3′, and 5′-TGA-3′. An x-ray crystal structure of Dpo4 was solved (2.1 Å) with a primer-template and A placed in the primer to be opposite the 1,N2-ϵ-G in the template 3′-(1,N2-ϵ-G)TACT 5′. The added A in the primer was paired across the template T with classic Watson-Crick geometry. Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar structure was observed with a ddGTP adjacent to the primer and opposite the C next to 1,N2-ϵ-G in 3′-(1,N2-ϵ-G)CACT-5′. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N2-ϵ-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations.

Efficient and High Fidelity Incorporation of Dctp Opposite 7,8-Dihydro-8-Oxodeoxyguanosine by Sulfolobus Solfataricus DNA Polymerase Dpo4

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed ≥95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured Kd,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The Ea for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca2+. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5′ C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.

Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase κ

DNA polymerase (pol) κ is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol κ, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol κ was studied with a series of eight N2-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH2(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, kcat/Km) were similar for insertion of dCTP opposite the lesions and for extension beyond the N2-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with kcat/Km values ∼10-3 less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (kp 5.9-10.3 s-1). The rapid bursts were abolished opposite BPG when α-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols η and ι; pol κ is the most resistant to N2-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N2-adducts, particularly the largest (N2-BPG).

Translesion Synthesis across O6-Alkylguanine DNA Adducts by Recombinant Human DNA Polymerases

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O6-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) δ also copied past these two lesions but was totally blocked by O6-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols ι and κ produced mainly only 1-base incorporation opposite O6-MeG and O6-BzG and had very low activity in copying O6-PobG. Human pol η copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O6-alkylG adducts for dCTP and dTTP with pol η and κ; pol ι showed a strong preference for dTTP. pol η, ι, and κ showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O6-MeG but little, if any, for O6-BzG or O6-PobG. Analysis of the pol η O6-PobG products indicated that the insertion of G was opposite the base (C) 5′ of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol η primer extension products indicated multiple components, mainly with C or T inserted opposite O6-alkylG but with no deletions in the cases of O6-MeG and O6-PobG. With pol η and O6-BzG, products were also obtained with –1 and –2 deletions and also with A inserted (opposite O6-BzG). The results with pol η may be relevant to some mutations previously reported with O6-alkylG adducts in mammalian cells.

Hydrogen Bonding of 7,8-Dihydro-8-Oxodeoxyguanosine with a Charged Residue in the Little Finger Domain Determines Miscoding Events in Sulfolobus Solfataricus DNA Polymerase Dpo4.

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a highly efficient and relatively accurate manner. Crystal structures have revealed a potential role for Arg332 in stabilizing the anti conformation of the 8-oxoG template base by means of a hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8-oxoG and dATP less favorable. Site-directed mutagenesis was used to replace Arg332 with Ala, Glu, Leu, or His in order to probe the importance of Arg332 in accurate and efficient bypass of 8-oxoG. The double mutant Ala331Ala332 was also prepared to address the contribution of Arg331. Transientstate kinetic results suggest that Glu332 retains fidelity against bypass of 8-oxoG that is similar to wild type Dpo4, a result that was confirmed by tandem mass spectrometric analysis of full-length extension products. A crystal structure of the Dpo4 Glu332 mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu332 and the O-8 atom of 8-oxoG. The space normally occupied by Arg332 side chain is empty in the crystal structures of the Ala332 mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu332 and Ala332 mutants. These results support the view that a bond between Arg332 and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.

Molecular Basis of Selectivity of Nucleoside Triphosphate Incorporation Opposite O6-Benzylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4 STEADY-STATE AND PRE-STEADY-STATE KINETICS AND X-RAY CRYSTALLOGRAPHY OF CORRECT AND INCORRECT PAIRING

Previous work has shown that Sulfolobus solfataricus DNA polymerase Dpo4-catalyzed bypass of O6-methylguanine (O6-MeG) proceeds largely in an accurate but inefficient manner with a “wobble” base pairing between C and O6-MeG (Eoff, R. L., Irimia, A., Egli, M., and Guengerich, F. P. (2007) J. Biol. Chem. 282, 1456–1467). We considered here the bulky lesion O6-benzylguanine (O6-BzG) in DNA and catalysis by Dpo4. Mass spectrometry analysis of polymerization products revealed that the enzyme bypasses and extends across from O6-BzG, with C the major product (∼70%) and some T and A (∼15% each) incorporated opposite the lesion. Steady-state kinetic parameters indicated that Dpo4 was 7-, 5-, and 27-fold more efficient at C incorporation opposite O6-BzG than T, A, or G, respectively. In transient state kinetic analysis, the catalytic efficiency was decreased 62-fold for C incorporation opposite O6-BzG relative to unmodified DNA. Crystal structures reveal wobble pairing between C and O6-BzG. Pseudo-“Watson-Crick” pairing was observed between T and O6-BzG. Two other structures illustrate a possible mechanism for the accommodation of a +1 frameshift in the Dpo4 active site. The overall effect of O6-BzG is to decrease the efficiency of bypass by roughly an order of magnitude in every case except correct bypass, where the effect is not as pronounced. By comparison, Dpo4 is more accurate but no more efficient than model replicative polymerases, such as bacteriophage T7- DNA polymerase and human immunodeficiency virus-1 reverse transcriptase in the polymerization past O6-MeG and O6-BzG.

Biochemical Basis of Genotoxicity of Heterocyclic Arylamine Food Mutagens HUMAN DNA POLYMERASE η SELECTIVELY PRODUCES A TWO-BASE DELETION IN COPYING THE N2-GUANYL ADDUCT OF 2-AMINO-3-METHYLIMIDAZO[4,5-f]QUINOLINE BUT NOT THE C8 ADDUCT AT THE NarI G3 SITE

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) η extended primers beyond template G-IQ adducts better than did pol κ and much better than pol ι or δ. In 1-base incorporation studies, pol η inserted C and A, pol ι inserted T, and pol κ inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol η. Mass spectrometry of pol η extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a –2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol η products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol η and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.

The C8-2'-deoxyguanosine adduct of 2-amino-3-methylimidazo[1,2-d]naphthalene, a carbocyclic analogue of the potent mutagen 2-amino-3-methylimidazo[4,5-f]quinoline, is a block to replication in vitro.

2-Amino-3-methylimidazo[1,2-d]naphthalene (cIQ) is a carbocyclic analogue of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in which a naphthalene ring system replaces the quinoline unit of IQ. The activity of cIQ in Ames Salmonella typhimurium tester strain TA98 is known to be 4-5 orders of magnitude lower than IQ. cIQ undergoes efficient bioactivation with rat liver microsomes. The C8-dGuo adduct was formed when calf thymus DNA was treated with the N-hydroxy-cIQ metabolite and either acetic anhydride or extracts from cells that overexpress N-acetyl transferase (NAT). These studies indicate that bioactivation, the stability of the N-hydroxylamine ester, and the reactivity of the nitrenium ion with DNA of cIQ are similar to IQ and that none of these factors account for the differences in mutagenic potency of these analogues in Ames assays. Oligonucleotides were synthesized that contain the C8-dGuo adduct of cIQ in the frameshift-prone CG-dinucleotide repeat unit of the NarI recognition sequence. We have examined the in vitro translesion synthesis of this adduct and have found it to be a strong replication block to Escherichia coli DNA polymerase I, Klenow fragment exo(-) (Kf(-)), E. coli DNA polymerase II exo(-) (pol II(-)), and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Previous studies by Fuchs and co-workers identified E. coli pol II as the polymerase responsible for two-base deletions of the C8-dGuo adduct of N-acetyl-2-aminofluorene in the NarI sequence. Our observation that pol II is strongly inhibited by the C8-dGuo adduct of cIQ suggests that one of the other SOS inducible polymerases (E. coli pol IV or pol V) is required for its bypass, and this accounts for the greatly attenuated mutagenicity in the Ames assays as compared with IQ.