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Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T(m) and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T(m) further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.
The DNA lesion 1,N(2)-ethenoguanine (1,N(2)-epsilon G) is formed endogenously as a by-product of lipid peroxidation or by reaction with epoxides that result from the metabolism of the industrial pollutant vinyl chloride, a known human carcinogen. DNA replication past 1,N(2)-epsilon G and site-specific mutagenesis studies on mammalian cells have established the highly mutagenic and genotoxic properties of the damaged base. However, there is as yet no information on the processing of this lesion during transcription. Here, we report the results of transcription past a site-specifically modified 1,N(2)-epsilon G DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen-bonding edge of guanine. Our results show that 1,N(2)-epsilon G acts as a partial block to the bacteriophage T7 RNA polymerase (RNAP), which allows nucleotide incorporation in the growing RNA with the selectivity A>G>(C=-1 deletion)>U. In contrast, 1,N(2)-epsilon G poses an absolute block to human RNAP II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNAP allows lesion bypass when the 1,N(2)-epsilon G adopts the syn-conformation. This orientation places the exocyclic ring in a collision-free empty pocket of the polymerase, and the observed base incorporation preferences are in agreement with hydrogen-bonding possibilities between the incoming nucleotides and the Hoogsteen edge of the lesion. On the other hand, in the more crowded active site of the human RNAP II, the modeling studies show that both syn- and anti-conformations of the 1,N(2)-epsilon G are sterically impermissible. Polymerase stalling is currently believed to trigger the transcription-coupled nucleotide excision repair machinery. Thus, our data suggest that this repair pathway is likely engaged in the clearance of the 1,N(2)-epsilon G from actively transcribed DNA.
A series of six oligonucleotides with dihydrodiol epoxide metabolites of the polycyclic aromatic hydrocarbons (PAHs) benz[a]anthracene and benzo[a]pyrene attached to adenine N6 and guanine N2 atoms were prepared and studied with the processive bacteriophage DNA polymerase T7, exonuclease- (T7-). HIV-1 reverse transcriptase was much less efficient in polymerization than T7-. Benz[a]anthracene and benzo[a]pyrene adducts strongly blocked incorporation of dTTP and dCTP opposite the A and G derivatives, respectively. dATP was preferentially incorporated in all cases. Steady state kinetic analysis indicated that the low catalytic efficiency with adducted DNA was due to both increased K(m) and lowered k(cat) values. Some differences due to PAH stereochemistry were observed. Fluorescence estimates of K(d) and presteady state kinetic measurements of k(off) showed no major decrease in the affinity of T7- with damaged DNA substrates or with dNTPs. Presteady state kinetics showed a lack of the normal burst kinetics for dNTP incorporation with all PAH-DNA derivatives. These results indicate that the rate-limiting step is at or before the step of phosphodiester bond formation; release of the oligonucleotide is no longer the slowest step. Thio elemental effects (substitution of alpha-oxygen with sulfur) were relatively small, in contrast to previous work with T7- and 8-oxo-7,8-dihydroguanine. The effect of these bulky PAH adducts is either to attenuate rates of conformational changes or to introduce an additional conformation problem but not to alter the inherent affinity of the polymerase for DNA or dNTPs.
Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7- (T7-) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts. dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased Km and attenuated kcat. Fluorescence estimates of Kd and pre-steady-state kinetic measurements of koff showed no significantly decreased affinity of T7- with the adducted oligonucleotides or the dNTP. Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme.DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always.
Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG.dA mismatch, thus providing insight into the high mutagenic potential of 8oG.
The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease(-) (T7(-)). RT and T7(-) effectively bypassed N(2)-methyl(Me)G and readily extended primers but were strongly blocked by N(2)-ethyl(Et)G, N(2)-isobutylG, N(2)-benzylG, and N(2)-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 10(3) in k(cat)/K(m) for dCTP incorporation opposite N(2)-MeG and a further large decrease opposite N(2)-EtG. Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another approximately 10(3)-fold by an Et group. dATP was preferentially incorporated opposite bulky N(2)-alkylG molecules. N(2)-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N(2)-EtG eliminated the bursts. Large elemental effects with thio-dCTP(alphaS) were observed with N(2)-EtG (6- and 72-fold decreases) but were much less with N(2)-MeG, indicating that the N(2)-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K(d(dCTP)) and K(d(DNA)) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.
The objective of this study was to target drug delivery to radiation-induced neoantigens, which include activated receptors within the tumor vasculature. These responses include posttranslational changes in pre-existing proteins, which can be discovered by phage-displayed peptide libraries administered to mice bearing irradiated tumors. Phage-displayed peptides recovered from irradiated tumors included the amino acid sequence RGDGSSV. This peptide binds to integrins within the tumor microvasculature. Immunohistochemical staining of irradiated tumors showed accumulation of fibrinogen receptor alpha(2b)beta(3) integrin. We studied tumor targeting efficiency of ligands to radiation-induced alpha(2b)beta(3). Radiopharmaceuticals were localized to irradiated tumors by use of alpha(2b)beta(3) ligands conjugated to nanoparticles and liposomes. Fibrinogen-conjugated nanoparticles bind to the radiation-activated receptor, obliterate tumor blood flow, and significantly increase regression and growth delay in irradiated tumors. Radiation-guided drug delivery to tumor blood vessels is a novel paradigm for targeted drug delivery.
The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 RT reverse transcriptase (HIV-1 RT), and bacteriophage T7 exo- (T7(-)) were examined to determine the misincorporation potential for 8-oxo-dGTP and to investigate the role of base pairing symmetry in DNA polymerase fidelity. 8-Oxo-dGTP was found to be a poor substrate for the four polymerases, with insertion efficiencies >10(4)-fold lower than for dGTP incorporation. Insertion efficiencies of 8-oxo-dGTP were also consistently lower than for incorporation of dNTPs opposite template 8-oxo-G, previously studied in this laboratory. In steady-state reactions, T7(-) had a high preference for 8-oxo-dGTP insertion opposite A (97%) and HIV-1 RT, KF-, and Pol II- preferred to insert 8-oxo-dGTP opposite C. Misinsertion frequencies for 8-oxo-dGTP also varied considerably from frequencies of misinsertion at template 8-oxo-G adducts for Pol II-, HIV-1 RT, and T7(-). Pre-steady-state incorporation of 8-oxo-dGTP opposite C (but not opposite A) by HIV-1 RT, KF-, and Pol II- displayed biphasic curves, with rates of initial incorporation 2- to 11-fold lower than normal dGTP incorporation. Although extension past template 8-oxo-G adducts had previously been shown to occur preferentially for the mispair, extension past primer 8-oxo-G:template A or C pairs was variable. The low and comparable estimated Kd values for dGTP and 8-oxo-dGTP binding to HIV-1 RT alone or HIV-1 RT.DNA complexes indicated that the initial binding was nonselective and had high affinity. The large difference (>3 orders of magnitude) in kinetic Kdapp values for 8-oxo-dGTP and dGTP binding to HIV-1 RT.DNA indicates that there are contributions to the kinetically determined Kdapp (such as conformational change and/or phosphodiester bond formation) which may be involved in the selection against 8-oxo-dGTP. The differences in binding (Kdapp), incorporation, and extension kinetics of 8-oxo-dGTP compared to normal dNTP incorporation at template 8-oxo-G adducts indicate that polymerase fidelity does not depend solely upon the overall geometry of Watson-Crick base pairs and reflects the asymmetry of the enzyme active site.
8-Oxo-7,8-dihydroguanine (8-oxoGua) can base pair with either cytosine (C) or adenine (A) when replicated by DNA polymerases. The 8-oxoGua.A mismatch is extended in preference to the 8-oxoGua.C pair. Using a model 25-mer/36-mer DNA duplex containing either guanine (Gua).C, 8-oxoGua.C, or 8-oxoGua.A base pairs at the primer terminus and A at the standing start position, we found that the pre-steady-state addition of dTTP opposite A following all three base pairs by bacteriophage T7 DNA polymerase exo- showed burst kinetics, suggesting that extension of all three base pairs is controlled by the rate of a step at or before phosphodiester bond formation. Substitution of dTTP alpha S for dTTP yielded modest thio effects of 1-6, suggesting that extension of all three pairs is limited by the rate of the conformational change prior to phosphodiester bond formation. Pre-steady-state values for kpol (maximum polymerization rate) were 120, 12, and 28 s-1, and Kd values were 2, 75, and 22 microM for insertion of dTTP following Gua.C, 8-oxoGua.C, and 8-oxoGua.A base pairs, respectively. Additional analysis of extension was provided by substitution of A in the standing start position by 2-aminopurine (2-AP), a fluorescent base analogue. Comparison of rapid-quench gel-based assays with stopped-flow fluorescence quenching assays suggested that during addition of dTTP opposite 2-AP phosphodiester bond formation was rate-limiting when 8-oxoGua.C or 8-oxoGua.A were the preceding base pairs, while conformational change was rate-limiting when Gua.C was the preceding base pair. Furthermore, the difference in apparent conformational change rates for addition of dTTP opposite 2-AP following the 8-oxoGua base pairs was greater than the differences in their phosphodiester bond formation rates, suggesting that discrimination in extension may be influenced more by conformational change rates than the rates of phosphodiester bond formation in this mispaired system.
Pre-steady-state kinetics of incorporation of dCTP and dATP opposite site-specific 8-oxo-7,8-dihydroguanine (8-oxoGua), in contrast to dCTP insertion opposite G, were examined as well as extension beyond the lesion using the replicative enzymes bacteriophage polymerase T7 exo- (T7-) and HIV-1 reverse transcriptase (RT). These results were compared to previous findings for Escherichia coli repair polymerases I (KF-) and II (pol II-) exo- [Lowe, L. G., & Guengerich, F. P. (1996) Biochemistry 35, 9840-9849]. HIV-1 RT showed a very high preference for insertion of dATP opposite 8-oxoGua, followed by pol II-, T7-, and KF-. Steady-state assays showed k(cat) consistently lower than pre-steady-state polymerization rates (k(p)) for insertion of dCTP opposite G or 8-oxoGua and insertion of dATP opposite 8-oxoGua. Pre-steady-state kinetic curves for the addition of dCTP opposite 8-oxoGua or G by KF-, pol II-, and T7- were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of dATP opposite 8-oxoGua by these polymerases did not display burst kinetics. With HIV-1 RT, addition of dATP opposite 8-oxoGua displayed burst kinetics while addition of dCTP did not. Analyses of the chemical step by substitution of phosphorothioate analogs for normal dNTPs suggest that the chemistry is rate-limiting during addition of dCTP and dATP opposite 8-oxoGua by KF-, pol II-, and T7-; HIV- RT did not show a chemical rate-limiting step during addition of dATP opposite 8-oxoGua. Kinetic assays performed with various dCTP concentrations indicate that dCTP has a higher Kd and lower k(p) for incorporation opposite 8-oxoGua compared to G with all four enzymes. The K(d,app)dATP values for KF-, pol II-, and T7- incorporation of dATP opposite 8-oxoGua, estimated in competition assays, were found to be 3-10-fold greater than the K(d)dCTP. Likewise, the K(d,app)dCTP for HIV-1 RT incorporation of dCTP opposite 8-oxoGua was found to be 10-fold greater than the K(d)dATP. The repair enzymes (KF- and pol II-) efficiently extended the 8-oxoGua x A pair; extension of 8-oxoGua x C was severely impaired, whereas the replicative enzymes (T7- and HIV-1 RT) extended both pairs, with faster rates for the extension of the 8-oxoGua x A pair. On the basis of these findings, the fidelity of all four enzymes during replication of 8-oxoGua depends on contributions from the apparent Kd, the ease of base pair extension, and either the rate of conformational change before chemistry or the rate of bond formation.