Jeong-Yun Choi

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 Abasic Lesions by Human B-Family and Y-Family DNA Polymerases α, δ, η, ι, κ, and REV1

Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides containing a synthetic abasic lesion (tetrahydrofuran analogue) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, respectively) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.

Kinetic Evidence for Inefficient and Error-prone Bypass across Bulky N2-Guanine DNA Adducts by Human DNA Polymerase ι

DNA polymerase (pol) ι has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol ι. Pol ι effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3–11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (Sp-2′-deoxycytidine 5′-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol η but not by pol ι is consistent with Hoogsteen base pairing by pol ι. Thus, polymerization by pol ι is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol ι may play a limited role in translesion synthesis on bulky N2-G adducts in cells.

Roles of the four DNA polymerases of the crenarchaeon Sulfolobus solfataricus and accessory proteins in DNA replication.

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3′ to 5′ exonuclease activities in vitro. Steady-state kinetic efficiencies (kcat/Km) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.

Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addition to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N2-alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH2(6-benzo[a]pyrenyl) (BP) and O6-alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O6-PobG, which caused almost complete blockage. Steady-state kinetic parameters (kcat/Km) were similar for insertion of dCTP opposite G and N2-G adducts but were severely reduced opposite the O6-G adducts. REV1 showed apparent pre-steady-state burst kinetics for dCTP incorporation only opposite N2-BPG and little, if any, opposite G, N2-benzyl (Bz)G, or O6-BzG. The maximal polymerization rate (kpol 0.9 s–1) opposite N2-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N2-BPG-adducted DNA 3-fold more tightly than unmodified G-containing DNA. These results and the lack of an elemental effect ((Sp)-2′-deoxycytidine 5′-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N2-BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N2-adduct at G but not to O6-adducts.

Human Rev1 polymerase disrupts G-quadruplex DNA

The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4–15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota.

O6-Methylguanine (O6-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O6-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O6-methylG. Misincorporation of dTTP opposite O6-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O6-methylG as the template base and incoming dCTP or dTTP were solved and showed that O6-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O6-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O6-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O6 atoms of O6-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O6-methylG by human pol ι, in contrast to the mispairing modes observed previously for O6-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

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.