Changes in metabolic landscapes shape divergent but distinct mutational signatures and cytotoxic consequences of redox stress.

Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress.

Tetrameric Acetyl-CoA Acetyltransferase 1 Is Important for Tumor Growth.

Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) regulates pyruvate dehydrogenase complex (PDC) by acetylating pyruvate dehydrogenase (PDH) and PDH phosphatase. How ACAT1 is "hijacked" to contribute to the Warburg effect in human cancer remains unclear. We found that active, tetrameric ACAT1 is commonly upregulated in cells stimulated by EGF and in diverse human cancer cells, where ACAT1 tetramers, but not monomers, are phosphorylated and stabilized by enhanced Y407 phosphorylation. Moreover, we identified arecoline hydrobromide (AH) as a covalent ACAT1 inhibitor that binds to and disrupts only ACAT1 tetramers. The resultant AH-bound ACAT1 monomers cannot reform tetramers. Inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation or AH treatment results in decreased ACAT1 activity, leading to increased PDC flux and oxidative phosphorylation with attenuated cancer cell proliferation and tumor growth. These findings provide a mechanistic understanding of how oncogenic events signal through distinct acetyltransferases to regulate cancer metabolism and suggest ACAT1 as an anti-cancer target.

Mutational signatures of redox stress in yeast single-strand DNA and of aging in human mitochondrial DNA share a common feature.

Redox stress is a major hallmark of cancer. Analysis of thousands of sequenced cancer exomes and whole genomes revealed distinct mutational signatures that can be attributed to specific sources of DNA lesions. Clustered mutations discovered in several cancer genomes were linked to single-strand DNA (ssDNA) intermediates in various processes of DNA metabolism. Previously, only one clustered mutational signature had been clearly associated with a subclass of ssDNA-specific apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. 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 predominance 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 supported the conclusion that hydrogen peroxide-induced mutagenesis is not the result of increased DNA polymerase misincorporation errors but rather is caused by direct damage to DNA. Proteins involved in modulation of chromatin status play a significant role in prevention of redox stress-induced mutagenesis, possibly by facilitating protection through modification of chromatin structure. These findings provide an opportunity for the search and identification of the mutational signature of redox stress in cancers and in other pathological conditions and could potentially be used for informing therapeutic decisions. In addition, the discovery of such signatures that may be present in related organisms should also advance our understanding of evolution.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

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 provide 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.

Evidence for Retromutagenesis as a Mechanism for Adaptive Mutation in Escherichia coli.

Adaptive mutation refers to the continuous outgrowth of new mutants from a non-dividing cell population during selection, in apparent violation of the neo-Darwinian principle that mutation precedes selection. One explanation is that of retromutagenesis, in which a DNA lesion causes a transcriptional mutation that yields a mutant protein, allowing escape from selection. This enables a round of DNA replication that establishes heritability. Because the model requires that gene expression precedes DNA replication, it predicts that during selection, new mutants will arise from damage only to the transcribed DNA strand. As a test, we used a lacZ amber mutant of Escherichia coli that can revert by nitrous acid-induced deamination of adenine residues on either strand of the TAG stop codon, each causing different DNA mutations. When stationary-phase, mutagenized cells were grown in rich broth before being plated on lactose-selective media, only non-transcribed strand mutations appeared in the revertants. This result was consistent with the known high sensitivity to deamination of the single-stranded DNA in a transcription bubble, and it provided an important control because it demonstrated that the genetic system we would use to detect transcribed-strand mutations could also detect a bias toward the non-transcribed strand. When residual lacZ transcription was blocked beforehand by catabolite repression, both strands were mutated about equally, but if revertants were selected immediately after nitrous acid exposure, transcribed-strand mutations predominated among the revertants, implicating retromutagenesis as the mechanism. This result was not affected by gene orientation. Retromutagenesis is apt to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counterselection rather than beforehand, and it may have roles in processes as diverse as the development of antibiotic resistance and neoplasia.

Mechanisms Regulating Protein Localization.

Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation.

Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd.

The current state of eukaryotic DNA base damage and repair.

DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair.

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. (56)iron and (28)silicon) by inhibiting Mre11 complex activity. Exposure of cells to (56)iron or (28)silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.