There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality.

Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

I and i

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore

PL02-05 

All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

Patterns of Somatic Mutation in Human Cancer Genomes

Nine transition metals were tested for the activation of three oxidants and the generation of inorganic radical species such as sulfate, peroxymonosulfate, and hydroxyl radicals. From the 27 combinations, 14 M/Ox couples demonstrated significant reactivity toward transforming a model organic substrate such as 2,4-dichlorophenol and are further discussed here. It was found that Co(II) and Ru(III) are the best metal catalysts for the activation of peroxymonosulfate. As expected on the basis of the Fenton reagent, Fe(III) and Fe(II) were the most efficient transition metals for the activation of hydrogen peroxide. Finally, Ag(I) showed the best results toward activating persulfate. Quenching studies with specific alcohols (tert-butyl alcohol and ethanol) were also performed to identify the primary radical species formed from the reactive M/Ox interactions. The determination of these transient species allowed us to postulate the rate-determining step of the redox reactions taking place when a metal is coupled with an oxidant in aqueous solution. It was found that when Co(II), Ru(III), and Fe(II) interact with peroxymonosulfate, freely diffusible sulfate radicals are the primary species formed. The same was proven for the interaction of Ag(I) with persulfate, but in this case caged or bound to the metal sulfate radicals might be formed as well. The conjunction of Ce(III), Mn(II), and Ni(II) with peroxymonosulfate showed also to generate caged or bound to the metal sulfate radicals. A combination of sulfate and hydroxyl radicals was formed from the conjunction of V(III) with peroxymonosulfate and from Fe(II) with persulfate. Finally, the conjunction of Fe(III), Fe(II), and Ru(III) with hydrogen peroxide led primarily to the generation of hydroxyl radicals. It is also suggested here that the redox behavior of a particular metal in solution cannot be predicted based exclusively on its size and charge. Additional phenomena such as metal hydrolysis as well as complexation with other counterions present in solution might affect the thermodynamics of the overall process and are further discussed here.

Radical generation by the interaction of transition metals with common oxidants.

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

ion phase that leads to an alkyl radical coordinated to the iron-hydroxo complex by a weak OH---C hydrogen bond, labeled as CI; (ii) an alkyl (or OH) rotation phase whereby the alkyl group achieves a favorable orientation for rebound; and (iii) a rebound phase that leads to C-O bond making and the ferric-alcohol complexes, 4,2P. The two profiles remain close in energy throughout the first two phases and then bifurcate. Whereas the HS state exhibits a significant barrier and a genuine TS for rebound, in the LS state, once the right orientation of the alkyl group is achieved, the LS rebound proceeds in a virtually barrier-free fashion to the alcohol. As such, alkane hydroxylation proceeds by TSR,70-72,120 in which the HS mechanism is truly stepwise with a finite lifetime for the radical intermediate, whereas the LS mechanism is effectively concerted with an ultrashort lifetime for the radical intermediate. Subsequent studies of ethane and camphor hydroxylation by the Yoshizawa group117,181-183 arrived at basically the same conclusion, that the mechanism is typified by TSR. The differences between the results of Shaik et al.130,173,177-180 and Yoshizawa et al.117,181-183 were rationalized recently71,72 and shown to arise owing to technical problems and the choice of the mercaptide ligand,117,181-183 which is a powerful electron donor and is too far from the representation of cysteine in the protein environment. The most recent study of camphor hydroxylation, which was done at a higher quality,117 converged to the picture reported by Shaik et al.130,173,177-180 and shows a stepwise HS process with a barrier of more than 3 kcal/mol for C-O bond formation by rebound of the camphoryl radical vis-à-vis an effectively concerted LS process for which this barrier is 0.7 kcal mol-1 and is the rotational barrier for reaching the rebound position. By referring to Figure 21, it is possible to rationalize the clock data of Newcomb in a simple manner. The apparent lifetimes are based on the assumption that there is a single state that leads to the reaction, such that the radical lifetime can be quantitated from the rate constant of free radical rearrangement and the ratio of rearranged to unrearranged alcohol product. However, in TSR, the rearranged (R) product is formed only/mainly on the HS surface, while the unrearranged (U) product is formed mainly on Figure 20. Formal descriptions of iron(III)-peroxo, iron(III)-hydroperoxo, and iron(V)-oxo species with indication of the negative charges. The roles “electrophile” or “nucleophile” are assigned according to the charge type. Reprinted with permission from ref 7. Copyright 2000 Springer-Verlag Heidelberg. 3964 Chemical Reviews, 2004, Vol. 104, No. 9 Meunier et al.

Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes.

Oxidative Nucleobase Modifications Leading to Strand Scission

Reactive oxygen species (ROS) have emerged as important cellular-signaling agents for cellular survival. Herein, we demonstrate that ROS-mediated oxidation of DNA to yield 8-oxo-7,8-dihydroguanine (OG) in gene promoters is a signaling agent for gene activation. Enhanced gene expression occurs when OG is formed in guanine-rich, potential G-quadruplex–forming sequences (PQS) in promoter-coding strands, initiating base excision repair (BER) by 8-oxoguanine DNA glycosylase (OGG1), yielding an abasic site (AP). The AP enables melting of the duplex to unmask the PQS, adopting a G-quadruplex fold in which apurinic/apyrimidinic endonuclease 1 (APE1) binds, but inefficiently cleaves, the AP for activation of vascular endothelial growth factor (VEGF) or endonuclease III-like protein 1 (NTHL1) genes. These details were mapped via synthesis of OG and AP analogs at single-nucleotide precision within the promoter of a luciferase reporter system. The reporters were analyzed in human and mouse cells while selectively knocking out or down critical BER proteins to identify the impact on luciferase expression. Identification of the oxidatively modified DNA base OG to guide BER activity in a gene promoter and impact cellular phenotype ascribes an epigenetic role to OG.

https://www.pnas.org/content/pnas/114/10/2604.full.pdf

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

Characterization of spiroiminodihydantoin as a product of one-electron oxidation of 8-Oxo-7,8-dihydroguanosine.

Single-stranded DNA genomes have been constructed that site-specifically contain the 7,8-dihydro-8-oxo-2‘-deoxyguanine (8-oxoG) oxidation products guanidinohydantoin (Gh) and the two stable stereoisomers of spiroiminodihydantoin (Sp1 and Sp2). The circular viral genomes were transfected into wild-type AB1157 Escherichia coli, and the efficiency of lesion bypass by DNA polymerase(s) was assessed. Viral progeny were analyzed for mutation frequency and type using the recently developed restriction endonuclease and postlabeling (REAP) assay. Gh was bypassed nearly as efficiently as the parent 8-oxoG but was highly mutagenic, causing almost exclusive G → C transversions. The stereoisomers Sp1 and Sp2 were, in comparison, much stronger blocks to DNA polymerase extension and caused a mixture of G → T and G → C transversions. The ratio of G → T to G → C mutations for each Sp lesion was dependent on the stereochemical configuration of the base. All observed mutation frequencies were at least an order of magnitude ...

The Hydantoin Lesions Formed from Oxidation of 7,8-Dihydro-8-oxoguanine Are Potent Sources of Replication Errors in Vivo†

Use of one-electron oxidants such as Na(2)IrCl(6) to oxidize 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) residues in oligodeoxynucleotides was previously shown to lead to predominant formation of a base lesion of mass M - 10 compared to starting material [Duarte et al. (1999) Nucleic Acids Res. 27, 596-502]. To thoroughly characterize the structure of this lesion, the oxidation of the nucleoside 9-N-(2',3',5'-tri-O-acetyl-beta-D-erythro-pentanosyl)-8-oxo-7,8-dihydroguanine with one-electron oxidants at pH 2-4 was used as a model for duplex DNA oxidation of OG residues. (1)H NMR and H,H COSY NMR studies in CD(3)OD along with LC-ESI-MS/MS fragmentation analysis are consistent with the assignment of the M - 10 species as a mixture of two pH-dependent equilibrating isomers, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) nucleoside, both present as mixtures of epimers at the C5 position of the hydantoin ring, i.e., four total isomers are formed. The Gh/Ia mixture is formed from hydration and decarboxylation of the initially formed intermediate 5-hydroxy-8-oxo-7,8-dihydroguanosine, a species that is also produced by four-electron oxidation (e.g., singlet oxygen) of guanosine. The product mixture can be further oxidized to a species designated Ia(ox), a hydrolytically unstable material at pH 7 that has been characterized by ESI-MS and (1)H NMR. Competition studies with 8-oxo-7,8-dihydroadenosine placed the redox potential of Gh/Ia at about 1.0 V vs NHE. These studies provide important information concerning the structures of lesions obtained when OG, a "hot spot" for oxidative damage, serves as a "hole trap" in long-range electron-transfer studies.

Cynthia J. Burrows

Papers

Oxidative Nucleobase Modifications Leading to Strand Scission

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

Characterization of spiroiminodihydantoin as a product of one-electron oxidation of 8-Oxo-7,8-dihydroguanosine.

The Hydantoin Lesions Formed from Oxidation of 7,8-Dihydro-8-oxoguanine Are Potent Sources of Replication Errors in Vivo†

Characterization of Hydantoin Products from One-Electron Oxidation of 8-Oxo-7,8-dihydroguanosine in a Nucleoside Model