Mechanisms of transcriptional and replicative mutagenesis

National Institute Of Environmental Health Sciences

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Abstract

The major objectives are to determine mechanisms of replicative (aim 1) and transcriptional (aim 2) mutagenesis relevant to the development of human disease (such as cancer) and to also determine the relationship between dysregulation of DNA repair pathways (i.e. base excision repair) and tumor development (aim 3). Aim 1 accomplishments. Redox stress is a major hallmark of cancer. Analysis of thousands of sequenced cancer exomes and whole genomes revealed distinct mutational signatures which can be attributed to specific sources of DNA lesions. Clustered mutations discovered in several cancer genomes reflect the persistence of single strand (ss)DNA intermediates arising as a result of various processes of DNA metabolism. Only one clustered mutation signature caused by a subclass of ssDNA-specific APOBEC cytidine deaminases was clearly defined so far. Others remain to be elucidated. We report here deciphering of the mutational spectra and mutational signature of redox stress in ssDNA of budding yeast and the signature of aging in human mitochondrial DNA. We found that the prevalence of C to T substitutions is a common feature of both signatures. Measurements of the frequencies of hydrogen peroxide-induced mutations in proofreading-defective yeast mutants allowed to conclude that hydrogen peroxide-induced mutagenesis is not the result of increased DNA polymerases misincorporation errors but rather is caused by direct damage to DNA. Proteins involved in modulation of chromatin status appeared to play a significant role in prevention of redox stress-induced mutagenesis, possibly by facilitating protection by modification of chromatin structure. These findings for the first time allow to enable the search for the mutational signature of redox stress in cancers and in other pathological conditions and could potentially be used for informing therapeutic decisions. In addition, such mutational signatures could be imprinted in evolutionary history of species and may further advance our understanding of mechanisms that drive evolution. Aim 2 accomplishments. P. aeruginosa is a Gram-negative, opportunistic pathogenic bacterium. It can cause acute infections in individuals with compromised immune systems, including cancer patients. It is the major pathogen associated with lung infections in cystic fibrosis patients. P. aeruginosa is the most common multidrug-resistant (MDR) Gram-negative pathogen causing pneumonia in hospitalized patients (36). According to the CDC, approximately 32,600 hospitalizations cases and 2,700 deaths associated with drug- resistant P. aeruginosa infections occurred in the US in 2017. (CDC. Antibiotic Resistance Threats in the United States, 2019. To address contribution of redox stress to acquisition of drug resistance in non-dividing, growth-stressed P. aeruginosa we exposed cells pre - grown in minimal media to several redox stress agents. While exposure to potassium bromate and hypochlorous acid led to an increase in frequencies of Cipro resistant (Cipro R) mutants compared to spontaneous mutation frequencies, hydrogen peroxide and S- nitrosoglutathione did not induce mutations even though they were applied in a wide range of doses, highlighting robust mechanisms of protection from redox stress in P. aeruginosa. For many years, mutations in the gyrA gene, coding for DNA gyrase, has been considered to be one of the main pathways of bacterial resistance to FQ (42). The majority of clinical strains of P. aeruginosa harbor such mutations (43). Contrary to these observations, Sanger sequencing of the gyrA gene of Cipro R mutants induced by potassium bromate and hypochlorous acid in stationary phase cultures unexpectedly revealed that the majority of these mutants do not carry mutation in gyrA. Whole genome sequencing (WGS) of Cipro R mutants uncovered several genes conferring Cipro resistance. Two thirds of all the mutations were mapped to spoT, N tRNA-synthetase and rpoN genes (Table 1). All of these genes are involved in the bacterial stringent response (SR). These findings suggest that non-dividing (or slow dividing) cells can acquire antibiotic resistance not by mutations modifying the mode of gyrase interaction with DNA in presence of Cipro, but by turning off the stress response, and thus may have important clinical implications. Aim 3 accomplishments (note: these studies were partly conducted at the NIEHS and partly conducted at Emory University (where Paul Doetsch was a faculty member prior to recruitment to the NIH). Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We also obtained evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer. During the recent year of support, we have established that overexpression of NTHL1 in both non-cancerous and human tumor cells confers sensitivity to the commonly employed anticancer agent, cisplatin. We also found novel protein interaction partners with NTHL1 and are exploiting this information to determine how other cell operational systems are impacted by NTHL1 dysregulation in the development of cancer, a major goal during the next fiscal year. We have also doscivered that NTHL1 overexpression sensitizes cells to the chemotherapeutic drug cisplatin. This fining has important implications for tumor cell responses to cisplatin therapy.