Synthesis of single and tandem DNA damages and mutagenesis studies of gamma-radiation-induced cross-linked guanine-thymine tandem lesion

The common environmental pollutant 1-nitropyrene and ionizing radiation are two examples of DNA damaging agents. Both these agents are mutagenic, tumorigenic, and carcinogenic. The reductive activation of the carcinogen 1-nitropyrene forms a C8 2\u27-deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG), and two N2 2\u27-deoxyguanosine adducts, 6-(deoxyguanosin-N2-yl)-1-aminopyrene ( N2-6-AP-dG) and 8-(deoxyguanosin-N 2-yl)-1-aminopyrene (N2-8-AP-dG), respectively. Ionizing radiation, on the other hand, gives rise to a large number of DNA damages, including tandem lesions such as a guanine-thymine, G[8,5-Me]T, and thymine-guanine, T[5-Me,8]G, intrastrand cross-links.^ To study the structural and biological effects of these DNA lesions, we used a total synthesis approach to synthesize the N2-AP-dG adducts and cross-links. The DNA adducts or cross-links were incorporated into defined sites of a dodecamer oligodeoxynucleotide, 5\u27-GTG CGTGTTTGT-3\u27, which contains the local DNA sequence (5\u27...CGT...3\u27) of the p53 codon 273. In the case of N2-AP-dG adducts, the modification was introduced in the dG residue of CG*T, whereas the cross-link was introduced in the GTG region (i.e., G^Tand T^G).^ Various conditions of Buchwald-Hartwig palladium-catalyzed amination have been examined to synthesize the two N2 2\u27-deoxyguanosine adducts. The most convenient synthetic approach involved a coupling between the protected 2\u27-deoxyguanosine and bromonitropyrenes, which, upon reductive deprotection, provided excellent yield of N 2-6-AP-dG and N2-8-AP-dG. The phosphoramidite derivatives of the N2-8- and N2-6-AP-dG adduct were prepared for solid-phase phosphoramidite oligodeoxynucleotide synthesis.^ 5-(Phenylthiomethyl)-2\u27-deoxyuridine is a photolabile precursor to the 5-(2\u27-deoxyuridilyl) methyl radical, which generates cross-link with adjacent guanine residues under anaerobic conditions. We synthesized oligodeoxynucleotides containing the cross-links by introducing 5-(phenylthiomethyl)-2\u27-deoxyuridine by total synthesis followed by UV-C irradiation of the purified oligodeoxynucleotide. The oligodeoxynucleotides containing the cross-links were characterized by using a series of analytical methods.^ The oligodeoxynucleotides containing the cross-links were incorporated into a plasmid vector, which was replicated in human embryonic kidney (293T/17) cells. Our results showed that both T[5-Me,8]G and G[8,5-Me]T lesions are mutagenic. Although the primary mutations induced by both cross-links were targeted G → T transversions, the T[5-Me,8]G cross-link was much more mutagenic than the G[8,5-Me]T cross-link (∼32% versus ∼18%). The targeted G → T transversions induced by T[5-Me,8]G (∼11%) was approximately 4 fold of that induced by G[8,5-Me]T (∼3%). In addition, in both cases semi-targeted mutations occurred at the 5\u27-adjacent base, which was mutated to T in significant frequency. To our knowledge, this is the first evidence of mutagenicity of these intra-strand cross-links in a cell.

Recognition and Incision of γ-Radiation-Induced Cross-Linked Guanine-Thymine Tandem Lesion G[8,5-Me]T by UvrABC Nuclease

Nucleotide excision repair (NER) plays an important role in maintaining the integrity of DNA by removing various types of bulky or distorting DNA adducts in both prokaryotic and eukaryotic cells. In Escherichia coli, the excision repair proteins UvrA, UvrB, and UvrC recognize and incise the bulky DNA damages induced by UV light and chemical carcinogens. In this process, when a putative lesion in DNA is identified initially by UvrA, a subsequent strand opening is carried out by UvrB that not only ensures that the distortion is indeed due to a damaged nucleotide but also recognizes the chemical structure of the modified nucleotides with varying efficiencies. UvrB also recruits UvrC that catalyzes both the 3′- and the 5′-incisions. Herein, we examined the interaction of UvrABC with a DNA substrate containing a single G[8,5-Me]T cross-link and compared it with T[6,4]T (the 6-4 pyrimidine-pyrimidone photoproduct) and the C8 guanine adduct of N-acetyl-2-aminofluorene (AAF). The intrastrand vicinal cross-link G[8,5-Me]T containing a covalent bond between the C8 position of guanine and the 5-methyl carbon of the 3′-thymine is formed by X-radiation, while T[6,4]T is a vicinal cross-link induced by the UV light. We also selected the AAF adduct for comparison because it represents a highly distorting monoadduct containing a covalent linkage at the C8 position of guanine. The dissociation constants (Kd) for UvrA protein binding to DNA substrates containing the G[8,5-Me]T, T[6,4]T, and AAF adducts, as determined by gel mobility shift assays, were 3.1 ± 1.3, 2.8 ± 0.9, and 8.2 ± 1.9, respectively. Although UvrA had a considerably higher affinity for G[8,5-Me]T than for the AAF adduct, the G[8,5-Me]T intrastrand cross-link was incised by UvrABC much less efficiently than the T[6,4]T intrastrand cross-link and the AAF adduct. Similar incision results also were obtained with the DNA substrates containing the adducts in a six-nucleotide bubble, indicating that the inefficient incision of G[8,5-Me]T cross-link by UvrABC was probably due to the lack of efficient recognition of the adduct by UvrB at the second step of DNA damage recognition in the E. coli NER. Indeed, as compared to T[6,4]T and AAF substrates, which clearly showed UvrB-DNA complex formation, very little UvrB complex was detectable with the G[8,5-Me]T substrate. Our result suggests that G[8,5-Me]T intrastrand cross-link is more resistant to excision repair in comparison with the T[6,4]T and AAF adducts and thus will likely persist longer in E. coli cells

Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η

Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G → T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5′ and three bases 3′ to the cross-link. The most prevalent semitargeted mutation was a C → T transition immediately 5′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G → T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5′ to the lesion was increased and 3′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determine if human polymerase η (hpol η) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol η can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol η was significantly different from that by yeast pol η in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol η opposite cross-linked T was 3−5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol η was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol η was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol η may play a role in correct bypass of the cross-links

Design, Synthesis, and Structure-Activity Relationships of Pyridoquinazolinecarboxamides as RNA Polymerase I Inhibitors

RNA polymerase I (Pol I) is a dedicated polymerase that transcribes the 45S ribosomal (r) RNA precursor. The 45S rRNA precursor is subsequently processed into the mature 5.8S, 18S, and 28S rRNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. On the basis of the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides have been evaluated as inhibitors of Pol I and activators of the destruction of RPA194, the Poll large catalytic subunit protein. Structure-activity relationships in assays of nucleolar stress and cell viability demonstrate key pharmacophores and their physicochemical properties required for potent activation of Pol I stress and cytotoxidty. This work identifies a set of bioactive compounds that potently cause RPA194 degradation that function in a tightly constrained chemical space. This work has yielded novel derivatives that contribute to the development of Pol I inhibitory cancer therapeutic strategies.Peer reviewe

Mechanism of Action Studies of Lomaiviticin A and the Monomeric Lomaiviticin Aglycon. Selective and Potent Activity Toward DNA Double-Strand Break Repair-Deficient Cell Lines

(−)-Lomaiviticin A (<b>1</b>) and the monomeric lomaiviticin aglycon [aka: (−)-MK7-206, (<b>3</b>)] are cytotoxic agents that induce double-strand breaks (DSBs) in DNA. Here we elucidate the cellular responses to these agents and identify synthetic lethal interactions with specific DNA repair factors. Toward this end, we first characterized the kinetics of DNA damage by <b>1</b> and <b>3</b> in human chronic myelogenous leukemia (K562) cells. DSBs are rapidly induced by <b>3</b>, reaching a maximum at 15 min post addition and are resolved within 4 h. By comparison, DSB production by <b>1</b> requires 2–4 h to achieve maximal values and >8 h to achieve resolution. As evidenced by an alkaline comet unwinding assay, <b>3</b> induces extensive DNA damage, suggesting that the observed DSBs arise from closely spaced single-strand breaks (SSBs). Both <b>1</b> and <b>3</b> induce ataxia telangiectasia mutated- (ATM-) and DNA-dependent protein kinase- (DNA-PK-) dependent production of phospho-SER139-histone H2AX (γH2AX) and generation of p53 binding protein 1 (53BP1) foci in K562 cells within 1 h of exposure, which is indicative of activation of nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. Both compounds also lead to ataxia telangiectasia and Rad3-related- (ATR-) dependent production of γH2AX at later time points (6 h post addition), which is indicative of replication stress. <b>3</b> is also shown to induce apoptosis. In accord with these data, <b>1</b> and <b>3</b> were found to be synthetic lethal with certain mutations in DNA DSB repair. <b>1</b> potently inhibits the growth of breast cancer type 2, early onset- (BRCA2-) deficient V79 Chinese hamster lung fibroblast cell line derivative (VC8), and phosphatase and tensin homologue deleted on chromosome ten- (PTEN-) deficient human glioblastoma (U251) cell lines, with LC₅₀ values of 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively, and selectivities of >11.6 versus the isogenic cell lines transfected with and expressing functional BRCA2 and PTEN genes. <b>3</b> inhibits the growth of the same cell lines with LC₅₀ values of 6.0 ± 0.5 and 11 ± 4 nM and selectivities of 84 and 5.1, for the BRCA2 and PTEN mutants, respectively. These data argue for the evaluation of these agents as treatments for tumors that are deficient in BRCA2 and PTEN, among other DSB repair factors

Specific and Efficient Binding of Xeroderma Pigmentosum Complementation Group A to Double-Strand/Single-Strand DNA Junctions With 3′- and/or 5′-SsDNA Branches

Human XPA is an important DNA damage recognition protein in nucleotide excision repair (NER). We previously observed that XPA binds to the DNA lesion as a homodimer [Liu, Y., Liu, Y., Yang, Z., Utzat, C., Wang, G., Basu, A. K., and Zou, Y. (2005) Biochemistry 44, 7361-7368]. Herein we report that XPA recognized undamaged DNA double-strand/single-strand (ds-ssDNA) junctions containing ssDNA branches with binding affinity (Kd = 49.1 ± 5.1 nM) much higher than its ability to bind to DNA damage. The recognized DNA junction structures include the Y-shape junction (with both 3′- and 5′-ssDNA branches), 3′-overhang junction (with a 3′-ssDNA branch), and 5′-overhang junction (with a 5′-ssDNA branch). Using gel filtration chromatography and gel mobility shift assays, we showed that the highly efficient binding appeared to be carried out by the XPA monomer and that the binding was largely independent of RPA. Furthermore, XPA efficiently bound to six-nucleotide mismatched DNA bubble substrates with or without DNA adducts including C8 guanine adducts of AF, AAF, and AP and the T[6,4]T photoproducts. Using a set of defined DNA substrates with varying degrees of DNA bending, we also found that the XPC-HR23B complex recognized DNA bending, whereas neither XPA nor the XPA-RPA complex could bind to bent DNA. We propose that, besides DNA damage recognition, XPA may also play a novel role in stabilizing, via its high affinity to ds-ssDNA junctions, the DNA strand opening surrounding the lesion for stable formation of preincision NER intermediates. Our results provide a plausible mechanistic interpretation for the indispensable requirement of XPA for both global genome and transcription-coupled repairs. Since ds-ssDNA junctions are common intermediates in many DNA metabolic pathways, the additional potential role of XPA in cellular processes is discussed. © 2006 American Chemical Society