A combination of direct reversion and nucleotide excision repair counters the mutagenic effects of DNA carboxymethylation.

Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O6-carboxymethylguanine (O6-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O6-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O6-CMG mutagenicity. To better address how cells detect and remove O6-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O6-CMG. We found that cells deficient in either the direct repair protein O6-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O6-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O6-CMG DNA damage.

Direct Alkylation of Deoxyguanosine by Azaserine Leads to O6-Carboxymethyldeoxyguanosine.

The O6-alkylguanosine adduct O6-carboxymethyldeoxyguanosine (O6-CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of O6-CMdG as well as the corresponding methyl adduct O6-methyldeoxyguanosine (O6-MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize O6-CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of O6-CMdG and O6-MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an O6-serine-CMdG (O6-Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O6-Ser-CMdG forms as an intermediate that spontaneously decomposes to form O6-CMdG. Finally, we quantified levels of O6-CMdG and O6-MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.

A Chemical Link between Meat Consumption and Colorectal Cancer Development?

O6-carboxymethylguanine (O6-CMG) is a mutagenic DNA adduct that forms at increased levels when people eat meat. It has been studied as a potential initiating event in colorectal carcinogenesis. It can arise from alkylation of guanine in DNA by electrophilic degradation products of N-nitroso compounds. There is significant data regarding biochemical and cellular process, including DNA repair and translesion DNA synthesis that control O6-CMG accumulation, persistence, and mutagenicity. Mutation spectra arising from the adduct closely resemble common mutations in colorectal cancer; however, gaps remain in understanding the biochemical processes that regulate how and where the damage persists in the genome. Addressing such questions relies on advances in chemistry such as synthesis approaches and bioanalytical methods. Results of research in this area help advance our understanding of the toxicological relevance of O6-CMG-modified DNA. Further attention should focus on understanding how a combination of genetic and environmental factors control its biological persistence and how this information can be used as a basis of biomoniotoring and prevention efforts to help mitigate colon cancer risk.

Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide.

DNA mutations can result from replication errors due to different forms of DNA damage, including low-abundance DNA adducts induced by reactions with electrophiles. The lack of strategies to measure DNA adducts within genomic loci, however, limits our understanding of chemical mutagenesis. The use of artificial nucleotides incorporated opposite DNA adducts by engineered DNA polymerases offers a potential basis for site-specific detection of DNA adducts, but the availability of effective artificial nucleotides that insert opposite DNA adducts is extremely limited, and furthermore, there has been no report of a quantitative strategy for determining how much DNA alkylation occurs in a sequence of interest. In this work, we synthesized an artificial nucleotide triphosphate that is selectively inserted opposite O6-carboxymethyl-guanine DNA by an engineered polymerase and is required for DNA synthesis past the adduct. We characterized the mechanism of this enzymatic process and demonstrated that the artificial nucleotide is a marker for the presence and location in the genome of O6-carboxymethyl-guanine. Finally, we established a mass spectrometric method for quantifying the incorporated artificial nucleotide and obtained a linear relationship with the amount of O6-carboxymethyl-guanine in the target sequence. In this work, we present a strategy to identify, locate, and quantify a mutagenic DNA adduct, advancing tools for linking DNA alkylation to mutagenesis and for detecting DNA adducts in genes as potential diagnostic biomarkers for cancer prevention.

DNA Adduct-Directed Synthetic Nucleosides.

Chemical damage to DNA is a key initiator of adverse biological consequences due to disruption of the faithful reading of the genetic code. For example, O6-alkylguanine ( O6-alkylG) DNA adducts are strongly miscoding during DNA replication when the damaged nucleobase is a template for polymerase-mediated translesion DNA synthesis. Thus, mutations derived from O6-alkylG adducts can have severe adverse effects on protein translation and function and are an early event in the initiation of carcinogenesis. However, the low abundance of these adducts places significant limitations on our ability to relate their presence and biological influences with resultant mutations or disease risk. As a consequence, there is a critical need for novel tools to detect and study the biological role of alkylation adducts. Incorporating DNA bases with altered structures that are derived synthetically is a strategy that has been used widely to interrogate biological processes involving DNA. Such synthetic nucleosides have contributed to our understanding of DNA structure, DNA polymerase (Pol) and repair enzyme function, and to the expansion of the genetic alphabet. This Account describes our efforts toward creating and applying synthetic nucleosides directed at DNA adducts. We synthesized a variety of nucleosides with altered base structures that complement the altered hydrogen bonding capacity and hydrophilicity of O6-alkylG adducts. The heterocyclic perimidinone-derived nucleoside Per was the first of such adduct-directed synthetic nucleosides; it specifically stabilized O6-benzylguanine ( O6-BnG) in a DNA duplex. Structural variants of Per were used to determine hydrogen bonding and base-stacking contributions to DNA duplex stability in templates containing O6-BnG as well as O6-methylguanine ( O6-MeG) adducts. We created synthetic probes able to stabilize damaged over undamaged templates and established how altered hydrogen bonding or base-stacking properties impact DNA duplex stability as a function of adduct structures. This knowledge was then applied to devise a hybridization-based detection strategy involving gold nanoparticles that distinguish damaged from undamaged DNA by colorimetric changes. Furthermore, synthetic nucleosides were used as mechanistic tools to understand chemical determinants such as hydrogen bonding, π-stacking, and size and shape deviations that impact the efficiency and fidelity of DNA adduct bypass by DNA Pols. Finally, we reported the first example of amplifying alkylated DNA, accomplished by combining an engineered polymerase and synthetic triphosphate for which incorporation is templated by a DNA adduct. The presence of the synthetic nucleoside in amplicons could serve as a marker for the presence and location of DNA damage at low levels in DNA strands. Adduct-directed synthetic nucleosides have opened new concepts to interrogate the levels, locations, and biological influences of DNA alkylation.

A gene-targeted polymerase-mediated strategy to identify O6-methylguanine damage.

Detecting DNA adducts in cancer genes is important for understanding cancer etiology. This study reports a strategy to identify the mutagenic DNA adduct O6-methylguanine in K-Ras. The strategy involves selective replication past a synthetic primer when placed opposite O6-methylguanine. Future work can apply this approach to other cancer-relevant genes.

Mechanism of RNA polymerase II stalling by DNA alkylation.

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

Modulation of Cytotoxicity by Transcription-Coupled Nucleotide Excision Repair Is Independent of the Requirement for Bioactivation of Acylfulvene.

Bioactivation as well as DNA repair affects the susceptibility of cancer cells to the action of DNA-alkylating chemotherapeutic drugs. However, information is limited with regard to the relative contributions of these processes to the biological outcome of metabolically activated DNA alkylating agents. We evaluated the influence of cellular bioactivation capacity and DNA repair on cytotoxicity of the DNA alkylating agent acylfulvene (AF). We compared the cytotoxicity and RNA synthesis inhibition by AF and its synthetic activated analogue iso-M0 in a panel of fibroblast cell lines with deficiencies in transcription-coupled (TC-NER) or global genome nucleotide excision repair (GG-NER). We related these data to the inherent bioactivation capacity of each cell type on the basis of mRNA levels. We demonstrated that specific inactivation of TC-NER by siRNA had the largest positive impact on AF activity in a cancer cell line. These findings establish that transcription-coupled DNA repair reduces cellular sensitivity to AF, independent of the requirement for bioactivation.