Termination sites of the in vitro DNA synthesis on single-stranded DNA photosensitized by promazines

Bacteriophage phi X174 and M13 mp9 single-stranded DNA molecules were primed either with restriction fragments or synthetic primers and irradiated with near UV light in the presence of promazine derivatives. These DNAs were used as template for in vitro complementary chain synthesis by Escherichia coli DNA polymerase I large fragment. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis of the synthesis products and localized by comparison with a standard dideoxy sequencing pattern. More than 90% of the chain terminations were mapped exactly one nucleotide before a guanine residue. In addition, photoreaction was shown to occur more predominantly with guanine residues localized in single-stranded parts of the genome. The same guanine residues could also be damaged when the reaction was performed, in the dark, in the presence of the artificially generated promazine cation radicals. Using the BamHI-SmaI adaptor (5'GATCCCCGGG-3'), it was shown that the guanine alteration was a covalent addition of the promazine, or of a cation radical photodegradation product, on the guanine moiety. Kinetics of chlorpromazine photoaddition on single-stranded and double-stranded DNAs were determined.     

Conversion of monofunctional DNA adducts of cis-diamminedichloroplatinum (II) to bifunctional lesions. Effect on the in vitro replication of single-stranded DNA by Escherichia coli DNA polymerase I and eukaryotic DNA polymerases alpha

Reaction of cis-diamminedichloroplatinum (II) with single-stranded M13 phage DNA in vitro produced monofunctional platinum-DNA adducts on guanine and bifunctional lesions with either two guanine bases (GG) or one adenine and one guanine (AG). When DNA containing a majority of monofunctional platinum-DNA lesions was dialyzed against 10 mM NaCIO4 at 37 degrees C, conversion of monoadducts to bifunctional lesions was observed. We examined the effect of post-treatment formation of bifunctional lesions on DNA synthesis by Escherichia coli DNA polymerase I and highly purified eukaryotic DNA polymerase alpha from Drosophila melanogaster and calf thymus. Arrest sites on the platinated template were determined by polyacrylamide gel electrophoresis. Monofunctional lesions did not appear to block DNA synthesis. Inhibition of replication increased as bifunctional platinum-DNA lesions formed during post-treatment incubation; GG adducts inhibited replication more than AG. These results suggest that bifunctional GG platinum-DNA adducts may be the major toxic damage of cisplatin.     

Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases

Hypoxanthine?xanthine oxidase?Fe3+?ethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA. The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol. Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands. When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced. This suggests that FapyAde, when present in DNA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase. FapyGua might possess similar properties.     

Effect of DNA base composition on the intercalation of proflavine. A kinetic study.

The effect of DNA base composition on the kinetics of the association between DNA and proflavine has been investigated using the temperature jump relaxation method. It is found that, regardless of the G + C base composition the results fit a two step mechanism, the second of which exhibits characteristics of intercalation of proflavine into DNA. However, they two equilibrium constants corresponding to these steps, KI and KII, depend on the nature of the DNAs. The constant KI is found to be an order of magnitude greater for M. lysodeikticus DNA (72% G + C) than for calf thymus DNA (48% G + C). Increasing G-C content thus appears to favor the intermediate non-intercalated complex of proflavine with DNA. Methylation of M. lysodeikticus DNA with dimethyl sulfate, preferentially yielding N7 methyl guanine as the modified base, again leads to an apparent two step mechanism, with the value of KI unchanged with respect to untreated DNA, while the affinity of proflavine for the intercalated complex measured by the value of KII increases for methylated DNA.     

Different effects of DNA adducts induced by carcinogenic and noncarcinogenic azo dyes on in vitro DNA synthesis

M13mp10 phage DNA modified with the carcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) or the noncarcinogen 2-methoxy-4-aminoazobenzene (2-MeO-AAB) was used as a template for E.coli DNA polymerase I. Analysis of the reaction products on DNA sequencing gels showed that with both types of compound the induced lesions blocked DNA synthesis, mainly at one base prior to guanine adducts, but that the inhibition by 3-MeO-AAB-adducts was substantially greater than that by 2-MeO-AAB-adducts. Thus different effects on DNA replication between 3-MeO-AAB- and 2-MeO-AAB-adducts might be a reflection of differences in their carcinogenic potency.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.     

Pyrosequencing for the quantitative assessment of 8-oxodG bypass DNA synthesis

Translesion synthesis (TLS) with specialized DNA polymerases allows dealing with a base lesion on the template strand during DNA replication; a base is inserted opposite the lesion, correctly or incorrectly, depending on the lesion, the involved DNA polymerase(s) and the sequence context. The major oxidized DNA base 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) is highly mutagenic due to its ability to pair with either cytosine or adenine during DNA synthesis, depending on its conformation and involved DNA polymerases. To measure the correct or mutagenic outcome of lesion bypass, an original quantitative pyrosequencing method was developed and analytically validated. The method was applied to the study of DNA synthesis fidelity through an 8-oxodG or an undamaged guanine. After an in vitro primer-extension through 8-oxodG in the presence of the four deoxynucleotides triphosphates and a total nuclear protein extract, obtained from normal human intestinal epithelial cells (FHs 74 Int cell line), the reaction products were amplified by polymerase chain reaction and analyzed by pyrosequencing to measure nucleotides inserted opposite the lesion. The 8-oxodG bypass fidelity of FHs 74 Int cells nuclear extract is about 85.3%. We calculated within-day and total precisions for both 8-oxodG (2.8% and 2.8%, respectively) and undamaged templates (1.0% and 1.1%, respectively). We also demonstrated that only cytosine is incorporated opposite a normal guanine and that both cytosine and adenine can be incorporated opposite an 8-oxodG lesion. The proposed method is straightforward, fast, reproducible and easily adaptable to other sequences and lesions. It thus has a wide range of applications in the biological field, notably to elucidate TLS mechanisms and modulators.     

Extrinsic cotton effects of aminoacridines bound to DNA

The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400–500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.     

Ring-opened 7-methylguanine residues in DNA are a block to in vitro DNA synthesis.

Single-stranded M13mp18 phage DNA was methylated with dimethylsulfate (DMS), and further treated with alkali to ring-open N7-methylguanine residues and yield 2-6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) residues. Nucleotide incorporation during in vitro DNA synthesis on methylated template using E. coli DNA polymerase Klenow fragment (Kf polymerase) was reduced compared to the unmethylated template. Additional treatment of the methylated template with NaOH to generate Fapy residues, further reduced in vitro DNA synthesis compared to the synthesis on methylated templates, which suggested that Fapy residues were a block to in vitro DNA synthesis. Analysis of the termination products on sequencing gels, assuming that synthesis stops one base before a blocking lesion, indicated that arrest of DNA synthesis upon direct alkylation of single-stranded DNA occurred 1 base 3' to template adenine residues in the case of Kf polymerase and 1 base 3' to adenine and cystosine residues for T4 polymerase. When the alkylated templates were treated with NaOH to produce a template which converted all the N7-methylguanine residues to Fapy residues, the blocks to DNA synthesis were still observed one base before adenine residues. In addition to the stops previously observed for the methylated templates, however, new stops occurred one base 3' to template guanine residues for synthesis using both Kf polymerase and T4 polymerase. Fapy residues, therefore, represent a potential lethal lesion which may also arrest in vivo DNA synthesis if not repaired.     

Studies on the structure of chemically methylated DNA

CD and melting temperature measurements on the nature of DNA with chemically methylated guanine-rich sites indicate that the stable secondary structure of DNA depicted by Ramstein et al- involves considerable distortions resulting from decreased base-base stacking interaction. Besides that quantum chemical data gained from PPP calculations are in favor of a weaker hydrogen bonding interaction in the methylated guanine-cytosine base pair. CD measurements demonstrate that methylated DNA-regions differ from the nonmethylated helical structure, since formation of a condensed conformation as occurs in the transition from B to the C-uke structure is prevented by positively charged methylated guanine residues. An increase in helix winding angle, however, can not be excluded. Binding ability of the dyes acridine orange, phenosafranine, and the antibiotic actinomycin C is lowered for methylated DNA, while binding of proflavine is, in accordance with the results of Ramstein and Leng, slightly enhanced. The reason for the opposite behavior of proflavine is at present not fully understood. In particular changes in the binding ability with dyes could not be correlated with base specificity of complex formation. It is discussed that structural changes in DNA towards a loose conformation decrease the binding tendency for acridine orange, phenosafranine, and actinomycin C.     

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

Despite nearly universal conservation through evolution, the precise function of the DinB/pol κ branch of the Y-family of DNA polymerases has remained unclear. Recent results suggest that DinB orthologs from all domains of life proficiently bypass replication blocking lesions that may be recalcitrant to DNA repair mechanisms. Like other translesion DNA polymerases, the error frequency of DinB and its orthologs is higher than the DNA polymerases that replicate the majority of the genome. However, recent results suggest that some Y-family polymerases, including DinB and pol κ, bypass certain types of DNA damage with greater proficiency than an undamaged template. Moreover, they do so relatively accurately. The ability to employ this mechanism to manage DNA damage may be especially important for types of DNA modification that elude repair mechanisms. For these lesions, translesion synthesis may represent a more important line of defense than for other types of DNA damage that are more easily dealt with by other more accurate mechanisms.     

Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases iota and kappa

DNA polymerase iota (Poliota) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Poliota in lesion bypass, however, has remained unclear. Poliota is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Poliota differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Poliota could incorporate nucleotides opposite the gamma-HOPdG adduct, which is formed from an initial reaction of acrolein with the N(2) of guanine. We show here that Poliota incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Polkappa, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Poliota and Polkappa is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.     

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.     

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.     

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.     

A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA

2-Hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon.     

Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.     

Importance of the O6 position of guanine residues in the binding of DNA methylase to DNA

Methylation of Micrococcus lysodeikticus DNA by purified DNA methylase isolated from L1210 leukaemia cells is potently and specifically inhibited by both hetero and homoribo and deoxyribopolynucleotides containing guanine residues. The inhibitory effect is unaffected by chain length, but is abolished when the O6 residue of guanine is substituted as in poly[d(O6MeG)]20. Potent inhibition is also shown by polyinosinic and polyxanthylic acids, but not by polyadenylic acid or by heteropolymers containing adenine and thymine. These results suggest that the 6-position of the purine nucleus is important in binding of the DNA methylase to a particular region of the DNA duplex and that the hydrogen bonding properties of this group are important in enzyme recognition.