Cyclohexene ring and Fjord region twist inversion in stereoisomeric DNA adducts of enantiomeric benzo[c]phenanthrene diol epoxides.

The sterically hindered, nonplanar fjord region polycyclic aromatic hydrocarbons (PAHs) have been of great interest because of the exceptionally high mutagenic and tumorigenic activity of certain of their metabolically activated diol epoxides. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region PAH, is metabolically activated to a pair of enantiomers, 1S,2R,3R,4S-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene, (+)-anti-B[c]PhDE, and the corresponding 1R,2S,3S,4R enantiomer, (-)-anti-B[c]PhDE. Both of these can bind covalently to the amino group of purines in DNA via trans addition. In the present work we carry out an extensive computational investigation of the 1R(+) and 1S(-)-trans-anti-B[c]Ph adducts to the base guanine, with the goal of delineating the conformational possibilities for the fjord region and the adjacent cyclohexene-type benzylic ring and their relevance to DNA duplexes. We created 10 369 starting structures for each adduct and minimized the energy using AMBER 5.0. A limited set of conformational families is computed, in which the R isomer structures are near mirror images of the S isomer. The benzylic rings are essentially all half-chair-type. Cyclohexene-type ring inversion as well as fjord region twist inversion are possible for each isomer and are correlated. DNA duplexes modified by fjord region adducts select conformers from the allowed families that optimize stacking interactions, which contributes to the stability of the carcinogen-intercalated DNA duplex structures [Cosman et al. (1993) Biochemistry 32, 12488-12497; Cosman et al. (1995) Biochemistry 34, 1295-1307; Suri et al. (1999) J. Mol. Biol. 292, 289-307; Lin et al. (2001) J. Mol. Biol. 306, 1059-1080]. In turn, this stability could contribute to the resistance to repair by the human nucleotide excision system observed in fjord region adducts [Buterin et al. (2000) Cancer Res. 60, 1849-1856].

Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.

The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus observed gel mobilities using classical reptation treatments requires enhancement of unmodified DNA flexibility in gels, compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.

Stereochemical, structural, and thermodynamic origins of stability differences between stereoisomeric benzo[a]pyrene diol epoxide deoxyadenosine adducts in a DNA mutational hot spot sequence.

Benzo[a]pyrene (BP), a prototype polycyclic aromatic hydrocarbon (PAH), can be metabolically activated to the enantiomeric benzo[a]pyrene diol epoxides (BPDEs), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the (-)-(7S,8R,9R,10S) enantiomer. These can react with adenine residues in DNA, to produce the stereoisomeric 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts. High-resolution NMR solution studies indicate that in DNA duplexes the 10R (-) adduct is intercalated on the 5'-side of the modified adenine, while the 10S (+) adduct is disordered, exhibits multiple adduct conformations, and is positioned on the 3'-side of the modified adenine. Duplexes containing the 10S (+) adduct positioned at A within codon 61 of the human N-ras sequence CAA are thermodynamically less stable and more easily excised by human DNA repair enzymes than those containing the 10R (-) adduct. However, the molecular origins of these differences are not understood and represent a fascinating opportunity for elucidating structure-function relationships. We have carried out a computational investigation to uncover the structural and thermodynamic origins of these effects in the 11-mer duplex sequence d(CGGACAAGAAG).d(CTTCTTGTCCG) by performing a 2-ns molecular dynamics simulation using NMR solution structures as the basis for the starting models. Then, we applied the MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method to compute free energy differences between the stereoisomeric adducts. The 10R (-) isomer is more stable by approximately 13 kcal/mol, of which approximately 10 kcal/mol is enthalpic, which agrees quite well with their observed differences in thermodynamic stability. The lower stability of the 10S (+) adduct is due to diminished stacking by the BP moiety in the intercalation pocket, more helix unwinding, and a diminished quality of Watson-Crick base pairing. The latter stems from conformational heterogeneity involving a syn-anti equilibrium of the glycosidic bond in the modified adenine residue. The lower stability and conformational heterogeneity of the 10S (+) adduct may play a role in its enhanced susceptibility to nucleotide excision repair.

Influence of bulky polynuclear carcinogen lesions in a TATA promoter sequence on TATA binding protein-DNA complex formation.

The TATA binding protein (TBP) is an essential component of the transcription initiation complex that recognizes and binds to the minor groove of the TATA DNA duplex consensus sequences. The objective of this study was to determine the effect of a carcinogen-modified adenine residue, positioned site-specifically within a regulatory TATA DNA sequence, on the binding of TBP. Two 25-mer oligonucleotides with stereoisomeric 10S (+)-trans-anti- or 10R (-)-trans-anti-BPDE-N(6)-dA residues at A(1) or A(2) within the TATA sequence element (5'-...TA(1)TAAA...-3')-(5'-...TTTA(2)TA...) were synthesized (anti-BPDE-N(6)-dA denotes an adduct formed from the reaction of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydobenzo[a]pyrene). The formation of complexes with TBP of these two sequences in the double-stranded forms (1 nM) were studied employing electrophoretic mobility shift assays (EMSA) at different TBP concentrations (0-70 nM). The overall affinity of TBP for the BPDE-modified target DNA sequences was weakly enhanced in the case of the (+)-trans or (-)-trans lesions positioned at site A(1) with K(d) approximately 8 and 6 nM, respectively (K(d) approximately 9 nM for the unmodified TATA DNA). Higher-order TBP-DNA complexes were observed at TBP concentrations in excess of approximately 15 nM. However, the stabilities of the biologically significant monomeric TBP-DNA complexes was dramatically increased or decreased, depending on the position of the lesion (A(1) or A(2)), or on its stereochemical and conformational characteristics. A molecular docking modeling approach was employed to insert the stereoisomeric BPDE residues into the known TATA box-TBP structure [Nikolov, D. B., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 4862-4867] to rationalize these observations. Native gel electrophoresis experiments with the same duplexes without TBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, nor that they differ from one another in this respect. These results suggest that the hydrophobic, bulky BPDE residues influence the binding of TBP by mechanisms other than prebending. The efficiency of RNA transcription of TBP-controlled promoters could be strongly influenced by the presence of such bulky lesions that could adversely affect the levels of gene expression.

Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I.

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.

Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo[c]phenanthrene diol epoxide DNA lesions.

Benzo[c]phenanthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo[c]phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE-DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (-)-trans-anti-B[c]PhDE-N(6)-dA lesion located within the sequence 5'-CTCTCACTTCC-3'. The results show that both (-)-trans-anti-B[c]PhDE-N(6)-dA and (+)-trans-anti-B[c]PhDE-N(6)-dA block RNA synthesis. Furthermore, both B[c]PhDE-dA stereoisomeric adducts lead to lower levels of initiation of transcription relative to that observed using an unmodified DNA template. In contrast to these results, placement of the adduct on the nontranscribed strand within the template does not impede transcription elongation. In addition to the assessment of the effect of the lesions on transcription elongation, the resulting transcripts were characterized in terms of their base composition. A high level of base misincorporation is detected at the 3'-ends of truncated transcripts, with guanosine being most frequently incorporated opposite the modified nucleotide rather than the expected uridine. This result supports the notion that translocation past a modified base in a DNA template relies in part on correct base incorporation, and suggests that stalling of RNA polymerases at damaged sites in DNA may well be dependent on both the presence of the lesion and the base which is incorporated opposite the modified nucleotide.

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides.

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.

Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair.

DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.

Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2'-deoxyguanosine but not of 2'-deoxyguanosine.

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2'-deoxyguanosine 5'-monophosphate (dGMP) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(-H)(*), derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO2(-)) to the nitrogen dioxide radical (*)NO2. In turn, we show that (*)NO2 can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The dGMP(-H)(*) neutral radicals formed via deprotonation of the dGMP(*)(+) radical cations and identified by their characteristic transient absorption spectrum (lambda(max) approximately 310 nm) oxidize nitrite anions with a rate constant of (2.6 +/- 0.3) x 10(6) M(-1) s(-1). The 8-oxo-dG is oxidized by (*)NO2 with a rate constant of (5.3 +/- 0.5) x 10(6) M(-1) s(-1). The 8-oxo-dG(-H)(*) neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (lambda(max) approximately 320 nm). The rate constant of 8-oxo-dG oxidation (k(12)) by the (*)NO2 one-electron oxidant (the (*)NO2/NO2(-) redox potential, E degrees approximately 1.04 V vs NHE) is lower than k(12) for a series of oxidizing aromatic radical cations with known redox potentials. The k(12) values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8-1.6 V versus NHE. The magnitude of k(12) gradually decreases from a value of 2.2 x 10(9) M(-1) s(-1) (E degrees = 1.62 V) to 5.8 x 10(8) M(-1) s(-1) (E degrees = 1.13 V) and eventually to 5 x 10(7) M(-1) s(-1) (E degrees = 0.91 V). The implications of these results, including the possibility that the redox cycling of the (*)NO2/NO2(-) species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.

Molecular topology of polycyclic aromatic carcinogens determines DNA adduct conformation: a link to tumorigenic activity.

We report below on the solution structures of stereoisomeric "fjord" region trans-anti-benzo[c]phenanthrene-N2-guanine (designated (BPh)G) adducts positioned opposite cytosine within the (C-(BPh)G-C).(G-C-G) sequence context. We observe intercalation of the phenanthrenyl ring with stereoisomer-dependent directionality, without disruption of the modified (BPh)G.C base-pair. Intercalation occurs to the 5' side of the modified strand for the 1S stereoisomeric adduct and to the 3' side for the 1R stereoisomeric adduct, with the S and R-trans-isomers related to one another by inversion in a mirror plane at all four chiral carbon atoms on the benzylic ring. Intercalation of the fjord region BPh ring into the helix without disruption of the modified base-pair is achieved through buckling of the (BPh)G.C base-pair, displacement of the linkage bond from the plane of the (BPh)G base, adaptation of a chair pucker by the BPh benzylic ring and the propeller-like deviation from planarity of the BPh phenanthrenyl ring. It is noteworthy that intercalation without base-pair disruption occurs from the minor groove side for S and R-trans-anti BPh-N2-guanine adducts opposite C, in contrast to our previous demonstration of intercalation without modified base-pair disruption from the major groove side for S and R-trans-anti BPh-N6-adenine adducts opposite T. Further, these results on fjord region 1S and 1R-trans-anti (BPh)G adducts positioned opposite C are in striking contrast to earlier research with "bay" region benzo[a]pyrene-N2-guanine (designated (BP)G) adducts positioned opposite cytosine, where 10S and 10R-trans-anti stereoisomers were positioned with opposite directionality in the minor groove without modified base-pair disruption. They also are in contrast to the 10S and 10R-cis-anti stereoisomers of (BP)G adducts opposite C, where the pyrenyl ring is intercalated into the helix with directionality, but the modified base and its partner on the opposite strand are displaced out of the helix. These results are especially significant given the known greater tumorigenic potential of fjord region compared to bay region polycyclic aromatic hydrocarbons. The tumorigenic potential has been linked to repair efficiency such that bay region adducts can be readily repaired while their fjord region counterparts are refractory to repair. Our structural results propose a link between DNA adduct conformation and repair-dependent mutagenic activity, which could ultimately translate into structure-dependent differences in tumorigenic activities. We propose that the fjord region minor groove-linked BPh-N2-guanine and major groove-linked BPh-N6-adenine adducts are refractory to repair based on our observations that the phenanthrenyl ring intercalates into the helix without modified base-pair disruption. The helix is therefore minimally perturbed and the phenanthrenyl ring is not available for recognition by the repair machinery. By contrast, the bay region BP-N2-G adducts are susceptible to repair, since the repair machinery can recognize either the pyrenyl ring positioned in the minor groove for the trans-anti groove-aligned stereoisomers, or the disrupted modified base-pair for the cis-anti base-displaced intercalated stereoisomers.

Error-prone lesion bypass by human DNA polymerase eta.

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase eta (Pol(eta)), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Pol(eta), we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Pol(eta) efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Pol(eta) effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant -1 deletion was also observed when the template base 5' to the abasic site is a T. Human Pol(eta) partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N:(2)-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in mammalian cells. These results show that human Pol(eta) is capable of error-prone translesion DNA syntheses in vitro and suggest that Pol(eta) may bypass certain lesions with a mutagenic consequence in humans.

The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli.

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)-anti-Benzo(a)pyrene diol epoxide [(+)-anti-BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G --> T transversions) and -1 frameshift mutations. The pathway leading to G --> T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.

Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures.

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.

Conformational determinants of structures in stereoisomeric cis-opened anti-benzo[a]pyrene diol epoxide adducts to adenine in DNA.

As part of a comprehensive effort to understand the origins of the variety of structural motifs adopted by (+)- and (-)-cis- and trans-anti-[BP]-N(2)-dG and -N(6)-dA adducts, with the goal of contributing to the elucidation of the structure-function relationship, we present results of our comprehensive computational investigation of the C10R (+)-cis- and C10S (-)-cis-anti-[BP]-N(6)-dA adducts on the nucleoside level. We have surveyed the potential energy surface of these two adducts by varying systematically, at 5 degrees intervals in combination, the three key torsion angle determinants of conformational flexibility (chi, alpha', and beta') in each adduct, creating 373 248 structures, and evaluating each of their energies. This has permitted us to map the entire potential energy surface of each adduct and to delineate the low-energy regions. The energy maps possess a symmetric relationship in the (+)/(-) adduct pair. This symmetry in the maps stems from the mirror image configuration of the benzylic rings in the two adducts, which produces opposite orientations of the BP residues in the C10R and C10S adducts on the nucleoside level. These opposite orientations result from primary steric hindrance between the base and the BP moiety which ensues when a (+) stereoisomer is rotated to the conformation favored by the (-) stereoisomer, and vice versa. Moreover, this steric hindrance manifested on the nucleoside level governs the structure on the duplex DNA level, accounting for observed opposite orientations in high-resolution NMR studies of C10R/C10S adduct pairs.

Accomodation of S-cis-tamoxifen-N(2)-guanine adduct within a bent and widened DNA minor groove.

The non-steroidal anti-estrogen tamoxifen [TAM] has been in clinical use over the last two decades as a potent adjunct chemotherapeutic agent for treatment of breast cancer. It has also been given prophylactically to women with a strong family history of breast cancer. However, tamoxifen treatment has also been associated with increased endometrial cancer, possibly resulting from the reaction of metabolically activated tamoxifen derivatives with cellular DNA. Such DNA adducts can be mutagenic and the activities of isomeric adducts may be conformation-dependent. We therefore investigated the high resolution NMR solution conformation of one covalent adduct (cis-isomer, S-epimer of [TAM]G) formed from the reaction of tamoxifen [TAM] to N(2)-of guanine in the d(C-[TAM]G-C).d(G-C-G) sequence context at the 11-mer oligonucleotide duplex level. Our NMR results establish that the S-cis [TAM]G lesion is accomodated within a widened minor groove without disruption of the Watson-Crick [TAM]G. C and flanking Watson-Crick G.C base-pairs. The helix axis of the bound DNA oligomer is bent by about 30 degrees and is directed away from the minor groove adduct site. The presence of such a bulky [TAM]G adduct with components of the TAM residue on both the 5'- and the 3'-side of the modified base could compromise the fidelity of the minor groove polymerase scanning machinery.

Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots.

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons display stronger tumorigenic activities in rodent studies than comparable bay region diol-epoxides, but the molecular basis for this difference between fjord and bay region derivatives is not understood. Here we tested whether the variable effects of these genotoxic metabolites of polycyclic aromatic hydrocarbons may result from different DNA repair reactions. In particular, we compared the repairability of DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but significantly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phenanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyclic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-induced proto-oncogene activation. Synthetic DNA substrates were assembled using a portion of human N-ras or H-ras that includes codon 61, and stereospecific B[a]P or B[c]Ph adducts were synthesized on adenine N6 at the second position of these two ras codon 61 sequences. DNA repair was determined by incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision products. These cell-free assays showed that all tested bay region B[a]P-N6-dA adducts are removed by the human nucleotide excision repair system, although excision efficiency varied with the particular stereochemical configuration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N6-dA adducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractory to the nucleotide excision repair process. These findings indicate that the exceptional tumorigenic potency of B[c]Ph or related fjord region diol-epoxides may be attributed, at least in part, to slow repair of the stable base adducts deriving from the reaction of these compounds with DNA.

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution.

The mechanism of decomposition of peroxynitrite (OONO(-)) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO(-) was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO(-), after protonation at pH 7.0 to HOONO, decomposes into (1)O(2) and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO(-) solution were added to a buffer solution (final pH 7.0-7.2), and the formation of (1)O(2) and NO(-) in high yields was observed. The (1)O(2) generated was trapped as the transannular peroxide (DPAO(2)) of 9, 10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO-) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO(-), the amount of singlet oxygen trapped was reduced by a factor of approximately 2 whereas the yield of trapping of NO(-) by methemoglobin remained unaffected. Because NO(3)(-) is known to be the ultimate decomposition product of OONO(-), these results suggest that the nitrate anion is not formed by a direct isomerization of OONO(-), but by an indirect route originating from NO(-).

Mismatch repair processing of carcinogen-DNA adducts triggers apoptosis.

The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSalpha and hMutSbeta efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.

Primer length dependence of binding of DNA polymerase I Klenow fragment to template-primer complexes containing site-specific bulky lesions.

The binding of the benzo[a]pyrene metabolite anti-BPDE (r7, t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) to the N(2) group of 2'-deoxyguanosine residues (dG) is known to adversely affect the Michaelis-Menten primer extension kinetics catalyzed by DNA Pol I and other polymerases. In this work, the impact of site-specific, anti-BPDE-modified DNA template strands on the formation of Pol I (Klenow fragment, KF)/template-primer complexes has been investigated. The 23-mer template strand 5'-d(AAC GC-(1) T(-)(2) ACC ATC CGA ATT CGC CC), I (dG = (+)-trans- and (-)-trans-anti-BPDE-N(2)-dG), was annealed with primer strands 18, 19, or 20 bases long. Complex formation of these template-primer strands with KF(-) (exonuclease-free) at different enzyme concentrations was determined using polyacrylamide gel mobility shift assays in the absence of dNTPs. The lesion dG causes an increase in the dissociation constants, K(d), of the monomeric, 1:1 KF(-)/DNA template-primer complexes by factors of 10-15 when the 3'-end base of the primer strand is positioned either opposite dG, or opposite dC(-)(1) in I, and the shapes of the binding isotherms are sigmoidal. The sigmoidal shapes are attributed to the formation of dimeric 2:1 KF(-)/DNA template-primer complexes. In contrast, when the 3'-end of the primer strand extends only to dT(-)(2) in I, the K(d) of 1:1 complexes is increased by factors of only 2-3, the shapes of the binding isotherms are hyperbolic and nonsigmoidal and are similar to those observed with the unmodified control, and monomeric KF(-)/DNA complexes are dominant. The impact of bulky lesions on polymerase/DNA complex formation in polymerase-catalyzed primer extension reactions needs to be taken into account in interpreting the site-specific Michaelis-Menten kinetics of these reactions.