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 K(d)(,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 E(a) 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 Ca(2+). 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.     

Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a thermostable archaeal enzyme and a member of the error-prone and lesion-bypass Y-family. In this paper, for the first time, the fidelity of a Y-family polymerase, Dpo4, was determined using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into an undamaged DNA substrate 21/41-mer at 37 degrees C. We assessed single-turnover (with Dpo4 in molar excess over DNA) saturation kinetics for all 16 possible nucleotide incorporations. The fidelity of Dpo4 was estimated to be in the range of 10(-3)-10(-4). Interestingly, the ground-state binding affinity of correct nucleotides (70-230 microM) is 10-50-fold weaker than those of replicative DNA polymerases. Such a low affinity is consistent with the lack of interactions between Dpo4 and the bound nucleotides as revealed in the crystal structure of Dpo4, DNA, and a matched nucleotide. The affinity of incorrect nucleotides for Dpo4 is approximately 2-10-fold weaker than that of correct nucleotides. Intriguingly, the mismatched dCTP has an affinity similar to that of the matched nucleotides when it is incorporated against a pyrimidine template base flanked by a 5'-template guanine. The incoming dCTP likely skips the first available template base and base pairs with the 5'-template guanine, as observed in the crystal structure of Dpo4, DNA, and a mismatched nucleotide. The mismatch incorporation rates, regardless of the 5'-template base, were approximately 2-3 orders of magnitude slower than the incorporation rates for matched nucleotides, which is the predominant contribution to the fidelity of Dpo4.     

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic polη

Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfataricus P2 reveal that it possesses a DinB homolog that has been termed DNA polymerase IV (Dpo4). We have overproduced and purified native Dpo4 protein and report here its enzymatic characterization. Dpo4 is thermostable, but can also synthesize DNA at 37°C. Under these conditions, the enzyme exhibits misinsertion fidelities in the range of 8 × 10^–3 to 3 × 10^–4. Dpo4 is distributive but at high enzyme to template ratios can synthesize long stretches of DNA and can substitute for Taq polymerase in PCR. On damaged DNA templates, Dpo4 can facilitate translesion replication of an abasic site, a cis-syn thymine–thymine dimer, as well as acetyl aminofluorene adducted- and cisplatinated-guanine residues. Thus, although phylogenetically related to DinB polymerases, our studies suggest that the archaeal Dpo4 enzyme exhibits lesion-bypass properties that are, in fact, more akin to those of eukaryotic polη.     

Mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV

The kinetic mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is resolved by pre-steady-state kinetic analysis of single-nucleotide (dTTP) incorporation into a DNA 21/41-mer. Like replicative DNA polymerases, Dpo4 utilizes an "induced-fit" mechanism to select correct incoming nucleotides. The affinity of DNA and a matched incoming nucleotide for Dpo4 was measured to be 10.6 nM and 230 microM, respectively. Dpo4 binds DNA with an affinity similar to that of replicative polymerases due to the presence of an atypical little finger domain and a highly charged tether that links this novel domain to its small thumb domain. On the basis of the elemental effect between the incorporations of dTTP and its thio analogue S(p)-dTTPalphaS, the incorporation of a correct incoming nucleotide by Dpo4 was shown to be limited by the protein conformational change step preceding the chemistry step. In contrast, the chemistry step limited the incorporation of an incorrect nucleotide. The measured dissociation rates of the enzyme.DNA binary complex (0.02-0.07 s(-1)), the enzyme.DNA.dNTP ternary complex (0.41 s(-1)), and the ternary complex after the protein conformational change (0.004 s(-1)) are significantly different and support the existence of a bona fide protein conformational change step. The rate-limiting protein conformational change was further substantiated by the observation of different reaction amplitudes between pulse-quench and pulse-chase experiments. Additionally, the processivity of Dpo4 was calculated to be 16 at 37 degrees C from analysis of a processive polymerization experiment. The structural basis for both the protein conformational change and the low processivity of Dpo4 was discussed.     

Insertion of dNTPs opposite the 1,N2-propanodeoxyguanosine adduct by Sulfolobus solfataricus P2 DNA polymerase IV

1, N (2)-Propanodeoxyguanosine (PdG) is a stable structural analogue for the 3-(2'-deoxy-beta- d- erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3 H)-one (M 1dG) adduct derived from exposure of DNA to base propenals and to malondialdehyde. The structures of ternary polymerase-DNA-dNTP complexes for three template-primer DNA sequences were determined, with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4), at resolutions between 2.4 and 2.7 A. Three template 18-mer-primer 13-mer sequences, 5'-d(TCACXAAATCCTTCCCCC)-3'.5'-d(GGGGGAAGGATTT)-3' (template I), 5'-d(TCACXGAATCCTTCCCCC)-3'.5'-d(GGGGGAAGGATTC)-3' (template II), and 5'-d(TCATXGAATCCTTCCCCC)-3'.5'-d(GGGGGAAGGATTC)-3' (template III), where X is PdG, were analyzed. With templates I and II, diffracting ternary complexes including dGTP were obtained. The dGTP did not pair with PdG, but instead with the 5'-neighboring template dC, utilizing Watson-Crick geometry. Replication bypass experiments with the template-primer 5'-TCACXAAATCCTTACGAGCATCGCCCCC-3'.5'-GGGGGCGATGCTCGTAAGGATTT-3', where X is PdG, which includes PdG in the 5'-CXA-3' template sequence as in template I, showed that the Dpo4 polymerase inserted dGTP and dATP when challenged by the PdG adduct. For template III, in which the template sequence was 5'-TXG-3', a diffracting ternary complex including dATP was obtained. The dATP did not pair with PdG, but instead with the 5'-neighboring T, utilizing Watson-Crick geometry. Thus, all three ternary complexes were of the "type II" structure described for ternary complexes with native DNA [Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001) Cell 107, 91-102]. The PdG adduct remained in the anti conformation about the glycosyl bond in each of these threee ternary complexes. These results provide insight into how -1 frameshift mutations might be generated for the PdG adduct, a structural model for the exocylic M 1dG adduct formed by malondialdehyde.     

Subtle but variable conformational rearrangements in the replication cycle of Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) may accommodate lesion bypass

The possible conformational changes of DNA polymerase IV (Dpo4) before and after the nucleotidyl-transfer reaction are investigated at the atomic level by dynamics simulations to gain insight into the mechanism of low-fidelity polymerases and identify slow and possibly critical steps. The absence of significant conformational changes in Dpo4 before chemistry when the incoming nucleotide is removed supports the notion that the "induced-fit" mechanism employed to interpret fidelity in some replicative and repair DNA polymerases does not exist in Dpo4. However, significant correlated movements in the little finger and finger domains, as well as DNA sliding and subtle catalytic-residue rearrangements, occur after the chemical reaction when both active-site metal ions are released. Subsequently, Dpo4's little finger grips the DNA through two arginine residues and pushes it forward. These metal ion correlated movements may define subtle, and possibly characteristic, conformational adjustments that operate in some Y-family polymerase members in lieu of the prominent subdomain motions required for catalytic cycling in other DNA polymerases like polymerase beta. Such subtle changes do not easily provide a tight fit for correct incoming substrates as in higher-fidelity polymerases, but introduce in low-fidelity polymerases different fidelity checks as well as the variable conformational-mobility potential required to bypass different lesions.     

A new anti conformation for N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) allows Watson-Crick pairing in the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)

Primer extension studies have shown that the Y-family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus P2 can preferentially insert C opposite N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) [F. Boudsocq, S. Iwai, F. Hanaoka and R. Woodgate (2001) Nucleic Acids Res., 29, 4607–4616]. Our goal is to elucidate on a structural level how AAF-dG can be harbored in the Dpo4 active site opposite an incoming dCTP, using molecular modeling and molecular dynamics simulations, since AAF-dG prefers the syn glycosidic torsion. Both anti and syn conformations of the templating AAF-dG in a Dpo4 ternary complex were investigated. All four dNTPs were studied. We found that an anti glycosidic torsion with C1′-exo deoxyribose conformation allows AAF-dG to be Watson–Crick hydrogen-bonded with dCTP with modest polymerase perturbation, but other nucleotides are more distorting. The AAF is situated in the Dpo4 major groove open pocket with fluorenyl rings 3′- and acetyl 5′-directed along the modified strand, irrespective of dNTP. With AAF-dG syn, the fluorenyl rings are in the small minor groove pocket and the active site region is highly distorted. The anti-AAF-dG conformation with C1′-exo sugar pucker can explain the preferential incorporation of dC by Dpo4. Possible relevance of our new major groove structure for AAF-dG to other polymerases, lesion repair and solution conformations are discussed.     

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,N(2)-Etheno(epsilon)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,N(2)-epsilon-G)TACT-5', among the four dNTPs tested individually. With the template 3'-(1,N(2)-epsilon-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,N(2)-epsilon-G)CACT-5') the major product was 5'-GTGA-3' and the minor product was 5'-AGTGA-3'. With the template 3'-(1,N(2)-epsilon-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 A) with a primer-template and A placed in the primer to be opposite the 1,N(2)-epsilon-G in the template 3'-(1,N(2)-epsilon-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,N(2)-epsilon-G in 3'-(1,N(2)-epsilon-G)CACT-5'. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N(2)-epsilon-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations.     

The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase eta and Sulfolobus solfataricus Dpo4

One of the most common DNA lesions arising in cells is an apurinic/apyrimidinic (AP) site resulting from base loss. Although a template strand AP site impedes DNA synthesis, translesion synthesis (TLS) DNA polymerases can bypass an AP site. Because this bypass is expected to be highly mutagenic because of loss of base coding potential, here we quantify the efficiency and the specificity of AP site bypass by two Y family TLS enzymes, Sulfolobus solfataricus DNA polymerase 4 (Dpo4) and human DNA polymerase eta (Pol eta). During a single cycle of processive DNA synthesis, Dpo4 and Pol eta bypass synthetic AP sites with 13-30 and 10-13%, respectively, of the bypass efficiency for undamaged bases in the same sequence contexts. These efficiencies are higher than for the A family, exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I. We then determined AP site bypass specificity for complete bypass, requiring insertion or misalignment at the AP site followed by multiple incorporations using the aberrant primer templates. Although Dpo4, Pol eta, and Klenow polymerase have different fidelity when copying undamaged DNA, bypass of AP sites lacking A or G by all three polymerases is nearly 100% mutagenic. The majority (70-80%) of bypass events made by all three polymerases are insertion of dAMP opposite the AP site. Single base deletion errors comprise 10-25% of bypass events, with other base insertions observed at lower rates. Given that mammalian cells contain five polymerases implicated in TLS, and given that a large number of AP sites are generated per mammalian cell per day, even moderately efficient AP site bypass could be a source of substitution and frameshift mutagenesis in vivo.     

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

We have investigated how a benzo[a]pyrene-derived N²-dG adduct, 10S(+)-trans-anti-[BP]-N²-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.     

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 Arg(332) 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 Arg(332) with Ala, Glu, Leu, or His in order to probe the importance of Arg(332) in accurate and efficient bypass of 8-oxoG. The double mutant Ala(331)Ala(332) was also prepared to address the contribution of Arg(331). Transientstate kinetic results suggest that Glu(332) 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 Glu(332) mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu(332) and the O-8 atom of 8-oxoG. The space normally occupied by Arg(332) side chain is empty in the crystal structures of the Ala(332) mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu(332) and Ala(332) mutants. These results support the view that a bond between Arg(332) and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.     

Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by "wobble" pairing between O6-methylguanine and cytosine, but accurate bypass is preferred

We examined the effect of a single O6-methylguanine (O6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O6-MeG, with C being the major product (approximately 70%) and T or A being the minor species (approximately 20% or approximately 10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that kpol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O6-MeG was approximately 6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O6-MeG. A structure containing T opposite O6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.     

Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation

The human DNA polymerase κ homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces “−1” frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N²-etheno-2′-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of −1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711–36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the “+1” base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of −1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for −1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed “dNTP-stabilized incorporation.” The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base.     

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question - what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? - has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N(2)-dG. In previous work, [+ta]-B[a]P-N(2)-dG was shown to be capable of inducing>95% G-->T mutations in one sequence context (5'-TGC), and approximately 95% G-->A mutations in another (5'-AGA). This raises the question - how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N(2)-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G-->T vs. G-->A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N(2)-dG and AP-site mutagenesis, as well as the so-called "A-rule".) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N(2)-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.     

Occurrence in the archaebacterium Sulfolobus solfataricus of a ribosomal protein complex corresponding to Escherichia coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)2.P0

Two-dimensional electrophoresis of total protein from 50 S ribosomal subunits of the archaebacterium Sulfolobus solfataricus demonstrated a complex between two proteins that was stable in 6 M urea, but dissociable in detergent or below pH 5.5. The proteins, numbered L1 and L10 according to their electrophoretic mobilities, corresponded to Escherichia coli ribosomal proteins L10 and L7/L12, respectively. The members of the complex were therefore designated Sso L10e and Sso L12e. Sso L12e had other properties in common with E. coli L7/L12: low molecular weight, relative acidity, selective release from the ribosome by high salt/ethanol, and dimeric structure. The Sso L12e.Sso L10e complex was isolated by gel filtration of total 50 S proteins in 4 M urea. The stoichiometry of the components was approximately four copies of Sso L12e to one copy of Sso L10e. The occurrence in an archaebacterium of a complex of acidic ribosomal proteins similar to E. coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)/.P0 strongly supports the concept that this element of quaternary structure is a major conserved feature of the ribosome and reaffirms its importance in the translocation step of protein synthesis.     

1H NMR study of self-association and restricted internal rotation of the C8-substituted deoxyguanosine 5'-monophosphate adduct of the carcinogen 2-(acetylamino)fluorene

The high-field 1H NMR spectra of a nucleotide-carcinogen adduct formed from 2-(acetylamino)fluorene (8-(N-fluoren-2-ylacetamido)-2'-deoxyguanosine 5'-monophosphate) have been examined in aqueous solution as a function of concentration at high and low temperatures. An anomalous concentration dependence of NMR spectra was observed at concentration levels over 1 mM. These spectral characteristics have been analyzed in terms of changes in self-association and in the interconversions between torsional diastereomers associated with the central nitrogen. Association constants have been computed. Stacking interactions, which involve both the fluorene and guanine rings, are strong, cooperative and highly temperature-dependent. Deacetylation alters the mode of stacking. Several effects of solvent and aggregation on the conformation at the central nitrogen are discussed.     

The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-5' exonuclease action of T4 DNA polymerase

When O-acetyl-4-(hydroxyamino)quinoline 1-oxide (Ac-4HAQO) reacts with double-stranded DNA at 37 degrees C the major products, N2-guanine, C8-guanine, and N6-adenine adducts, are formed in the proportions of 5:3:2, respectively. When the reaction is carried out with single-stranded DNA at 0 degree C, the products are found in the ratio 1:7:2. Unique 174-bp DNA fragments were modified in these ways and used as substrates for the 3'-5' exonuclease activity of T4 DNA polymerase. The results obtained showed that the exonuclease is blocked by the N2-guanine adduct but not the other two adducts. Interpretation of the cleavage patterns suggested that the enzyme stopped 2 nucleotides before the N2-guanine adduct. The N2-guanine adduct lies in the minor groove of the DNA double helix, while the other two adducts are found in the major groove. Apparently, only the former hinders progression of the enzyme.     

Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.