John M. Essigmann

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC-->AT transitions and GC-->TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to assess the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase η (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD′2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase κ, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10–15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the ‘steric gate’ residue of DinB (Phe13 → Val) and that of its archaeal homologue Dbh (Phe12 → Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.

The mismatch-repair protein hMSH2 binds selectively to DNA adducts of the anticancer drug cisplatin

BACKGROUND The antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) exerts its cytotoxic effects through the formation of covalent DNA adducts. A family of proteins possessing a common HMG box motif that binds specifically to cisplatin DNA adducts has been previously suggested to be important in the clinical efficacy of the drug. RESULTS We have shown that the human mismatch-repair protein, hMSH2, also binds specifically to DNA containing cisplatin adducts and displays selectivity for the DNA adducts of therapeutically active platinum complexes. Moreover, hMSH2 is overexpressed in testicular and ovarian tissue; tumors in these tissues are most effectively treated by cisplatin. CONCLUSIONS Our results suggest a role for hMSH2 in mediating cisplatin toxicity. Supporting this view, previous studies in Escherichia coli dam- strains demonstrate that mutations in mismatch-repair proteins confer resistance to cisplatin toxicity. Mismatch-repair deficiency is also correlated with tolerance to O6-methylguanine, a cytotoxic DNA lesion formed by methylating agents. A current model ascribes O6-methylguanine toxicity to unsuccessful attempts at repair of this lesion by mismatch-repair proteins, resulting in a futile cycle of incision and synthesis, leading ultimately to lethal DNA-strand breaks. We propose that mismatch repair may contribute to cisplatin toxicity by a similar mechanism. Alternatively, hMSH2 may shield cisplatin adducts from repair, allowing adducts to persist, thus enhancing lethality.

AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo.

Oxidative stress converts lipids into DNA-damaging agents. The genomic lesions formed include 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC), in which two carbons of the lipid alkyl chain form an exocyclic adduct with a DNA base. Here we show that the newly characterized enzyme AlkB repairs εA and εC. The potent toxicity and mutagenicity of εA in Escherichia coli lacking AlkB was reversed in AlkB+ cells; AlkB also mitigated the effects of εC. In vitro, AlkB cleaved the lipid-derived alkyl chain from DNA, causing εA and εC to revert to adenine and cytosine, respectively. Biochemically, εA is epoxidized at the etheno bond. The epoxide is putatively hydrolyzed to a glycol, and the glycol moiety is released as glyoxal. These reactions show a previously unrecognized chemical versatility of AlkB. In mammals, the corresponding AlkB homologs may defend against aging, cancer and oxidative stress.

The aflatoxin B1 formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma

A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B1 (AFB1). Hypotheses have been put forth that AFB1, in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB1 is 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB1-formamidopyrimidine (AFB1-FAPY) adduct. AFB1-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB1 exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AFB1-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB1-N7-Gua, and (ii) one proposed rotamer of AFB1-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.

Multiple pathways of recombination define cellular responses to cisplatin

BACKGROUND Cisplatin is a DNA-damaging drug used for treatment of testicular tumors. The toxicity of cisplatin probably results from its ability to form DNA adducts that inhibit polymerases. Blocked replication represents a particular challenge for tumor cells, which are committed to unremitting division. Recombination provides a mechanism by which replication can proceed despite the presence of lesions and therefore could be significant for managing cisplatin toxicity. RESULTS Recombination-deficient Escherichia coli mutants were strikingly sensitive to cisplatin when compared with the parental strain. Our data identified both daughter-strand gap and double-strand break recombination pathways as critical for survival following treatment with cisplatin. Although it is established that nucleotide excision repair (NER) significantly protects against cisplatin toxicity, most recombination-deficient strains were as sensitive to the drug as the NER-deficient uvrA mutant. Recombination/NER deficient double mutants were more sensitive to cisplatin than the corresponding single mutants, suggesting that recombination and NER pathways play independent roles in countering cisplatin toxicity. Cisplatin was a potent recombinogen in comparison with the trans isomer and canonical alkylating agents. Mitomycin C, which like cisplatin, forms DNA cross-links, was also recombinogenic at minimally toxic doses. CONCLUSIONS We have demonstrated that all of the major recombination pathways are critical for E. coli survival following treatment with cisplatin. Moreover, recombination pathways act independently of NER and are of equal importance to NER as genoprotective systems against cisplatin toxicity. Taken together, these results shed new light on how cells survive and succumb to this widely used anticancer drug.

Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes

Nanomaterials are often functionalized with biological ligands to enable their use as sensors of biological activity. However, the intricacies of nano-bio interactions are poorly understood, which hampers our ability to design nanomaterial-based sensors. Current experimental tools have been unable to visualize interactions occurring on the nano-bio interface with the spatial and temporal resolution needed to quantify biological interactions at their fundamental length and time scales. To fill the need for concurrent visualization of nanoparticles and biomolecules, we have combined two common microscopy techniques, one being for the study of biomolecules and the other for the study of nanoparticles, into a single instrument that has the capacity to study both nanoparticles and biological molecules simultaneously with spatial and temporal resolution that is appropriate for nanoscale interactions. This novel instrument has been used for the characterization of high-sensitivity sensors by designing synthetic biological polymers to selectively encapsulate single-wall carbon nanotubes. The design of synthetic sensing tools based on nanoparticle-biomolecule hybrids is promising for areas in need of high-specificity sensors, such as label-free detection of molecules within a cell, nanoparticle-based diagnostic tools, and nanoscale therapeutics. We introduce three examples of high-sensitivity and high-selectivity synthetic sensors that have the ability to detect a variety of molecules on a single-molecule scale: riboflavin, L-thyroxine, and oestradiol. These sensors have been used to detect and quantify riboflavin levels within a live murine macrophage cell in real-time. The findings provided herein will enable the development of early-onset diagnostic tools at the level of a single cell.

INHIBITION OF RNA POLYMERASE II TRANSCRIPTION IN HUMAN CELL EXTRACTS BY CISPLATIN DNA DAMAGE

The anticancer drug cisplatin induces a spectrum of lesions in DNA. The effect of such DNA damage on transcription by RNA polymerase II (RNA pol II) in human cell extracts was investigated at the level of initiation and elongation. RNA pol II transcription directed from the adenovirus major late promoter was inhibited following treatment of the promoter-containing template with increasing concentrations of cisplatin. Furthermore, transcription from an undamaged promoter fragment was depleted in the presence of increasing amounts of cisplatin DNA damage on an exogenous plasmid, suggesting such damage may hijack an essential factor for transcription initiation. The effect of cisplatin damage on RNA pol II elongation was investigated using site-specifically-placed cisplatin adducts. The GTG adduct was an effective block to RNA pol II elongation, inhibiting the polymerase by 80%. In contrast, RNA pol II completely bypassed the cisplatin GG intrastrand adduct. These studies suggest that the inhibition of RNA pol II transcription observed following the treatment of cells with cisplatin is likely to reflect the combined effects of DNA damage at the level of both transcription initiation and elongation.

Mutation of HIV-1 Genomes in a Clinical Population Treated with the Mutagenic Nucleoside KP1461

The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. Here we present an extensive analysis of viral sequences from HIV-1 infected volunteers from the first “mechanism validation” phase II clinical trial of a mutagenic base analog in which individuals previously treated with antiviral drugs received 1600 mg of KP1461 twice per day for 124 days. Plasma viral loads were not reduced, and overall levels of viral mutation were not increased during this short-term study, however, the mutation spectrum of HIV was altered. A large number (N = 105 per sample) of sequences were analyzed, each derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated control individuals (>7.1 million base pairs of unique viral templates were sequenced). We found that private mutations, those not found in more than one viral sequence and likely to have occurred in the most recent rounds of replication, increased in treated individuals relative to controls after 56 (p = 0.038) and 124 (p = 0.002) days of drug treatment. The spectrum of mutations observed in the treated group showed an excess of A to G and G to A mutations (p = 0.01), and to a lesser extent T to C and C to T mutations (p = 0.09), as predicted by the mechanism of action of the drug. These results validate the proposed mechanism of action in humans and should spur development of this novel antiretroviral approach.

Recognition and Processing of a New Repertoire of DNA Substrates by Human 3-Methyladenine DNA Glycosylase (AAG)

The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N(6)-ethenoadenine (epsilonA). The crystal structures of AAG bound to epsilonA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In this study, we explore the substrate specificity of full-length and truncated Delta80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions but excised only a few of them. Single-turnover excision kinetics showed that in addition to the well-known epsilonA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with epsilonA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N(2)-ethenoguanine (1,N(2)-epsilonG), albeit weakly, from duplex DNA. Uracil was excised from both single- and double-stranded DNA, but only by full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on double-stranded DNA, AAG excised both epsilonA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.