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.

[1]  P. Steinberg.  Red Meat-Derived Nitroso Compounds, Lipid Peroxidation Products and Colorectal Cancer , 2019, Foods.

[2]  Hongyuan Chen,et al.  Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O6 -Carboxymethyl Guanine and Reveals Its Behavior in Replication. , 2019, Angewandte Chemie.

[3]  Yinsheng Wang,et al.  Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells , 2019, The Journal of Biological Chemistry.

[4]  Hongyuan Chen,et al.  Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O 6 ‐Carboxymethyl Guanine and Reveals Its Behavior in Replication , 2019, Angewandte Chemie.

[5]  Hailey L Gahlon,et al.  DNA Adduct-Directed Synthetic Nucleosides. , 2019, Accounts of chemical research.

[6]  Hailey L Gahlon,et al.  A gene-targeted polymerase-mediated strategy to identify O6-methylguanine damage. , 2019, Chemical communications.

[7]  Dennis G. Gillingham,et al.  High Sensitivity of Human Translesion DNA Synthesis Polymerase κ to Variation in O6-Carboxymethylguanine Structures. , 2019, ACS chemical biology.

[8]  J. Chaput,et al.  Evolution of a General RNA-Cleaving FANA Enzyme , 2018, Nature Communications.

[9]  Yinsheng Wang,et al.  Cytotoxic and mutagenic properties of O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli cells , 2018, The Journal of Biological Chemistry.

[10]  Daniel B. Sloan,et al.  Detecting Rare Mutations and DNA Damage with Sequencing-Based Methods. , 2018, Trends in biotechnology.

[11]  Junzhou Wu,et al.  Nucleotide-Resolution Genome-Wide Mapping of Oxidative DNA Damage by Click-Code-Seq. , 2018, Journal of the American Chemical Society.

[12]  Ryan W. Kung,et al.  Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O 6 -Alkylguanine Adducts , 2018, Helvetica Chimica Acta.

[13]  T. Nohmi,et al.  Chemically-Induced DNA Damage, Mutagenesis, and Cancer , 2018, International journal of molecular sciences.

[14]  G. Margison,et al.  O6-methylguanine–induced transcriptional mutagenesis reduces p53 tumor-suppressor function , 2018, Proceedings of the National Academy of Sciences.

[15]  K. Diederichs,et al.  Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase. , 2017, Chemical communications.

[16]  S. Adar,et al.  Dynamic maps of UV damage formation and repair for the human genome , 2017, Proceedings of the National Academy of Sciences.

[17]  S. Adar,et al.  Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene , 2017, Proceedings of the National Academy of Sciences.

[18]  Z. Suo,et al.  Replication studies of carboxymethylated DNA lesions in human cells , 2017, Nucleic acids research.

[19]  S. Sturla,et al.  DNA Adducts from Anticancer Drugs as Candidate Predictive Markers for Precision Medicine , 2016, Chemical research in toxicology.

[20]  Chuan He,et al.  Base-Resolution Analysis of Cisplatin-DNA Adducts at the Genome Scale. , 2016, Angewandte Chemie.

[21]  S. Sturla,et al.  Bypass of Mutagenic O(6)-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases. , 2016, Chemical research in toxicology.

[22]  L. Vanhaecke,et al.  In vitro DNA adduct profiling to mechanistically link red meat consumption to colon cancer promotion. , 2016, Toxicology research.

[23]  Yinsheng Wang,et al.  Quantification of Azaserine-Induced Carboxymethylated and Methylated DNA Lesions in Cells by Nanoflow Liquid Chromatography-Nanoelectrospray Ionization Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method. , 2016, Analytical chemistry.

[24]  A. Marx,et al.  The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts , 2016, Nucleic acids research.

[25]  S. Sturla,et al.  In-Gene Quantification of O(6)-Methylguanine with Elongated Nucleoside Analogues on Gold Nanoprobes. , 2016, Journal of the American Chemical Society.

[26]  Fritz Eichenseher,et al.  Specific incorporation of an artificial nucleotide opposite a mutagenic DNA adduct by a DNA polymerase. , 2015, Journal of the American Chemical Society.

[27]  T. van de Wiele,et al.  O⁶-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat. , 2014, Molecular nutrition & food research.

[28]  D. Sargent,et al.  KRAS Codon 12 and 13 Mutations in Relation to Disease-Free Survival in BRAF–Wild-Type Stage III Colon Cancers from an Adjuvant Chemotherapy Trial (N0147 Alliance) , 2014, Clinical Cancer Research.

[29]  N. Sahgal,et al.  Development of a liquid chromatography/tandem mass spectrometry method to investigate the presence of biomarkers of DNA damage in urine related to red meat consumption and risk of colorectal cancer. , 2013, Rapid communications in mass spectrometry : RCM.

[30]  Hailey L Gahlon,et al.  Hydrogen bonding or stacking interactions in differentiating duplex stability in oligonucleotides containing synthetic nucleoside probes for alkylated DNA. , 2013, Chemistry.

[31]  Z. Wawrzak,et al.  Recognition of O6-benzyl-2′-deoxyguanosine by a perimidinone-derived synthetic nucleoside: a DNA interstrand stacking interaction , 2013, Nucleic acids research.

[32]  N. Tretyakova,et al.  Quantitation of DNA adducts by stable isotope dilution mass spectrometry. , 2012, Chemical research in toxicology.

[33]  F. Pasmans,et al.  An approach based on ultra-high pressure liquid chromatography-tandem mass spectrometry to quantify O6-methyl and O6-carboxymethylguanine DNA adducts in intestinal cell lines. , 2012, Journal of chromatography. A.

[34]  Daniela C Dieterich,et al.  Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition. , 2010, Journal of the American Chemical Society.

[35]  G. Maga,et al.  DNA Polymerases: Discovery, Characterization and Functions in Cellular DNA Transactions , 2010 .

[36]  C. Gloeckner,et al.  Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries , 2010, Current protocols in chemical biology.

[37]  R. Sinha,et al.  A large prospective study of meat consumption and colorectal cancer risk: an investigation of potential mechanisms underlying this association. , 2010, Cancer research.

[38]  T. Sugimura,et al.  Detection of endogenous DNA adducts, O6‐carboxymethyl‐2′‐deoxyguanosine and 3‐ethanesulfonic acid‐2′‐deoxycytidine, in the rat stomach after duodenal reflux , 2008, Cancer science.

[39]  C. Gloeckner,et al.  Evolving a thermostable DNA polymerase that amplifies from highly damaged templates. , 2007, Angewandte Chemie.

[40]  S. Sturla,et al.  A synthetic nucleoside probe that discerns a DNA adduct from unmodified DNA. , 2007, Journal of the American Chemical Society.

[41]  M. Weller,et al.  Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine , 2007, Oncogene.

[42]  G. Scott,et al.  Potassium diazoacetate-induced p53 mutations in vitro in relation to formation of O6-carboxymethyl- and O6-methyl-2'-deoxyguanosine DNA adducts: relevance for gastrointestinal cancer. , 2006, Carcinogenesis.

[43]  Richard Bowman,et al.  Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk. , 2006, Cancer research.

[44]  B. Cupid,et al.  Detection of O6-carboxymethyl-2'-deoxyguanosine in DNA following reaction of nitric oxide with glycine and in human blood DNA using a quantitative immunoslot blot assay. , 2004, Chemical research in toxicology.

[45]  G. Margison,et al.  Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine. , 2002, Mutagenesis.

[46]  D. Cooper,et al.  Detection of concomitant formation of O6-carboxymethyl- and O6-methyl-2'-deoxyguanosine in DNA exposed to nitrosated glycine derivatives using a combined immunoaffinity/HPLC method. , 1999, Chemical research in toxicology.

[47]  A. Pegg,et al.  Alkylation of DNA in rat tissues following administration of streptozotocin. , 1981, Cancer research.

[48]  R. Montesano.  Alkylation of DNA and tissue specificity in nitrosamine carcinogenesis. , 1981, Journal of supramolecular structure and cellular biochemistry.