Methods to Study Trinucleotide Repeat Instability Induced by DNA Damage and Repair.

Trinucleotide repeat (TNR) instability (expansion and deletion) is associated with more than 42 human neurodegenerative diseases and cancer and mediated by DNA replication, repair, recombination, and gene transcription. Somatic TNR instability is involved in the progression of TNR expansion diseases and can be modulated by DNA damage repair and gene transcription. Recent studies from our group and others have shown that DNA base damage and its repair play an active role in modulating TNR instability and are responsible for somatic age-dependent CAG repeat expansion in neurons of Huntington's disease mice induced by oxidative DNA damage. However, it remains to be elucidated how DNA damage, non-B form DNA structures, and DNA repair enzymes and cofactors can coordinate to regulate somatic TNR instability. Understanding the molecular mechanisms underlying DNA damage and repair-mediated somatic TNR instability is critically important for identification of new therapeutic targets for treatment and prevention of TNR-related diseases. Here we describe the methods to study the locations and distribution of DNA base lesions and their effects on TNR instability through DNA base excision repair in in vitro reconstituted human systems.

Electron-Mediated Aminyl and Iminyl Radicals from C5 Azido-Modified Pyrimidine Nucleosides Augment Radiation Damage to Cancer Cells.

Two classes of azido-modified pyrimidine nucleosides were synthesized as potential radiosensitizers; one class is 5-azidomethyl-2'-deoxyuridine (AmdU) and cytidine (AmdC), while the second class is 5-(1-azidovinyl)-2'-deoxyuridine (AvdU) and cytidine (AvdC). The addition of radiation-produced electrons to C5-azido nucleosides leads to the formation of π-aminyl radicals followed by facile conversion to σ-iminyl radicals either via a bimolecular reaction involving intermediate α-azidoalkyl radicals in AmdU/AmdC or by tautomerization in AvdU/AvdC. AmdU demonstrates effective radiosensitization in EMT6 tumor cells.

Pyrimidine Nucleosides with a Reactive (ß-Chlorovinyl)sulfone or (ß-Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA.

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(ß-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary ß-sulfonylvinylamines gave 5-(ß-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5'-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions.

Modulation of trinucleotide repeat instability by DNA polymerase ß polymorphic variant R137Q.

Trinucleotide repeat (TNR) instability is associated with human neurodegenerative diseases and cancer. Recent studies have pointed out that DNA base excision repair (BER) mediated by DNA polymerase ß (pol ß) plays a crucial role in governing somatic TNR instability in a damage-location dependent manner. It has been shown that the activities and function of BER enzymes and cofactors can be modulated by their polymorphic variations. This could alter the function of BER in regulating TNR instability. However, the roles of BER polymorphism in modulating TNR instability remain to be elucidated. A previous study has shown that a pol ß polymorphic variant, polßR137Q is associated with cancer due to its impaired polymerase activity and its deficiency in interacting with a BER cofactor, proliferating cell nuclear antigen (PCNA). In this study, we have studied the effect of the pol ßR137Q variant on TNR instability. We showed that pol ßR137Q exhibited weak DNA synthesis activity to cause TNR deletion during BER. We demonstrated that similar to wild-type pol ß, the weak DNA synthesis activity of pol ßR137Q allowed it to skip over a small loop formed on the template strand, thereby facilitating TNR deletion during BER. Our results further suggest that carriers with pol ßR137Q polymorphic variant may not exhibit an elevated risk of developing human diseases that are associated with TNR instability.

Arsenic trioxide co-exposure potentiates benzo(a)pyrene genotoxicity by enhancing the oxidative stress in human lung adenocarcinoma cell.

Although both arsenic trioxide (As2O3) and benzo(a)pyrene (BaP) are well-established human carcinogens, the interaction between As2O3 and BaP is synergistic or antagonistic remains controversial in terms of the existing studies. In addition, the mechanisms responsible for the combined effects are still unclear. In this study, we examined the potential interactive effects between As2O3 (1, 5, and 10 µM) and BaP (5, 10, and 20 µM) in cultured A549 cells by treating with BaP and As2O3 alone or in combination at various concentrations for 24 h. The single and combined effects of As2O3 and BaP on the cytotoxicity, DNA/chromosomal damage, and oxidative stress were examined by using tetrazolium (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye colorimetric assay, colony formation assay, fluorescence probe, chemical colorimetry, comet assay as well as micronucleus test. Our results showed that As2O3 synergistically enhanced the cytotoxicity, genotoxicity, and level of oxidative stress induced by BaP at various tested concentrations. Also, our experimental results showed that intracellular glutathione (GSH) contents were increased by various doses of BaP, but single or cotreatment with As2O3 significantly decreased the GSH level in the cells at all tested concentrations. Taken together, our results suggest that As2O3 may exert its synergistic cyto- and genotoxic effects with BaP mainly via elevated intracellular reactive oxygen species and reduced GSH contents and superoxide dismutase activities, thus promoting high level of oxidative stress, which may be a pivotal mechanism underlying As2O3 cocarcinogenic action.

DNA polymerase beta is involved in the protection against the cytotoxicity and genotoxicity of cigarette smoke.

Reactive oxygen species (ROS) and oxidative DNA damage have been implicated in the cigarette smoke-induced cytotoxicity and genotoxicity. DNA polymerase ß (polß), a key base excision repair (BER) enzyme in repairing oxidative DNA damage, may play a crucial role in fighting against the cytotoxicity and genotoxicity of cigarette smoke. In this study, we applied a novel approach to collect cigarette smoke extract (CSE) and investigated the cytotoxic and genotoxic effects of CSE by using the mouse embryo fibroblasts that express wild-type of polß (polß(+/+)), null of polß (polß(-/-)) and overexpression of polß (polß(oe)). Our results showed that polß(-/-) cells treated with CSE exhibited a higher ROS level and more DNA single-strand breaks and chromosomal aberrations than that of polß(+/+) and polß(oe) cells. These data suggested that polß mediated-BER may involve in repairing the CSE-induced DNA damage and protection against the cytotoxicity and genotoxicity of CSE.