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.

DNA damaging agents are a constant threat to genomes in both the nucleus and the mitochondria. To combat this threat, a suite of DNA repair pathways cooperate to repair numerous types of DNA damage. If left unrepaired, these damages can result in the accumulation of mutations which can lead to deleterious consequences including cancer and neurodegenerative disorders. The base excision repair (BER) pathway is highly conserved from bacteria to humans and is primarily responsible for the removal and subsequent repair of toxic and mutagenic oxidative DNA lesions. Although the biochemical steps that occur in the BER pathway have been well defined, little is known about how the BER machinery is regulated. The budding yeast, Saccharomyces cerevisiae is a powerful model system to biochemically and genetically dissect BER. BER is initiated by DNA N-glycosylases, such as S. cerevisiae Ntg1. Previous work demonstrates that Ntg1 is post-translationally modified by SUMO in response to oxidative DNA damage suggesting that this modification could modulate the function of Ntg1. In this study, we mapped the specific sites of SUMO modification within Ntg1 and identified the enzymes responsible for sumoylating/desumoylating Ntg1. Using a non-sumoylatable version of Ntg1, ntg1ΔSUMO, we performed an initial assessment of the functional impact of Ntg1 SUMO modification in the cellular response to DNA damage. Finally, we demonstrate that, similar to Ntg1, the human homologue of Ntg1, NTHL1, can also be SUMO-modified in response to oxidative stress. Our results suggest that SUMO modification of BER proteins could be a conserved mechanism to coordinate cellular responses to DNA damage.

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.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Mammalian cells are constantly and unavoidably exposed to DNA damage from endogenous and exogenous sources, frequently to the detriment of genomic integrity and biological function. Cells acquire a large number of chemically diverse lesions per day, and each can have a different genetic fate and biological consequences. However, our knowledge of how and when specific lesions are repaired or how they may compromise the fidelity of DNA replication or transcription and lead to deleterious biological endpoints in mammalian cells is limited. Studying individual lesions requires technically challenging approaches for the targeted introduction of defined lesions into relevant DNA sequences of interest. Here, we present a systematic analysis of factors influencing yield and an improved, efficient and reliable protocol for the production of mammalian expression phagemid vectors containing defined DNA base modifications in any sequence position of either complementary DNA strand. We applied our improved protocol to study the transcriptional mutagenesis-mediated phenotypic consequences of the common oxidative lesion 5-hydroxyuracil, placed in the G12 mutational hotspot of the KRAS oncogene. 5-OHU induced sustained oncogenic signaling in Neil1 Neil1-/-Neil2 -/- mouse cells. The resulting advance in technology will have broad applicability for investigation of single lesion DNA repair, mutagenesis, and DNA damage responses in mammalian cells.

DNA replication stress is an inefficient DNA synthesis process that leads replication forks to progress slowly or stall. Two main factors that cause replication stress are alterations in pools of deoxyribonucleotide (dNTP) precursors required for DNA synthesis and changes in the activity of proteins required for synthesis of dNTPs. Ribonucleotide reductase (RNR), containing regulatory hRRM1 and catalytic hRRM2 subunits, is the enzyme that catalyzes the conversion of ribonucleoside diphosphates (NDP) to deoxyribonucleoside diphosphates (dNDP) and thereby provides dNTP precursors needed for the synthesis of DNA. Here, we demonstrate that either endogenous or exogenous expression of Bcl2 results in decreases in RNR activity and intracellular dNTP, retardation of DNA replication fork progression, and increased rate of fork asymmetry leading to DNA replication stress. Bcl2 colocalizes with hRRM1 and hRRM2 in the cytoplasm and directly interacts via its BH4 domain with hRRM2 but not hRRM1. Removal of the BH4 domain of Bcl2 abrogates its inhibitory effects on RNR activity, dNTP pool level, and DNA replication. Intriguingly, Bcl2 directly inhibits RNR activity by disrupting the functional hRRM1/hRRM2 complex via its BH4 domain. Our findings argue that Bcl2 reduces intracellular dNTPs by inhibiting ribonucleotide reductase activity, thereby providing insight into how Bcl2 triggers DNA replication stress.

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates (dNDPs) to provide dNTP precursors for DNA synthesis. Here, we report that acetylation and deacetylation of the RRM2 subunit of RNR acts as a molecular switch that impacts RNR activity, dNTP synthesis, and DNA replication fork progression. Acetylation of RRM2 at K95 abrogates RNR activity by disrupting its homodimer assembly. RRM2 is directly acetylated by KAT7, and deacetylated by Sirt2, respectively. Sirt2, which level peak in S phase, sustains RNR activity at or above a threshold level required for dNTPs synthesis. We also find that radiation or camptothecin-induced DNA damage promotes RRM2 deacetylation by enhancing Sirt2–RRM2 interaction. Acetylation of RRM2 at K95 results in the reduction of the dNTP pool, DNA replication fork stalling, and the suppression of tumor cell growth in vitro and in vivo. This study therefore identifies acetylation as a regulatory mechanism governing RNR activity.

Localized hyper-mutability caused by accumulation of lesions in persistent single-stranded (ss) DNA has been recently found in several types of cancers. An increase in endogenous levels of reactive oxygen species (ROS) is considered to be one of the hallmarks of cancers. Employing a yeast model system, we addressed the role of oxidative stress as a potential source of hyper-mutability in ssDNA by modulation of the endogenous ROS levels and by exposing cells to oxidative DNA-damaging agents. We report here that under oxidative stress conditions the majority of base substitution mutations in ssDNA are caused by erroneous, DNA polymerase (Pol) zeta-independent bypass of cytosines, resulting in C to T transitions. For all other DNA bases Pol zeta is essential for ROS-induced mutagenesis. The density of ROS-induced mutations in ssDNA is lower, compared to that caused by UV and MMS, which suggests that ssDNA could be actively protected from oxidative damage. These findings have important implications for understanding mechanisms of oxidative mutagenesis, and could be applied to development of anticancer therapies and cancer prevention.

DNA base damage and non-coding apurinic/apyrimidinic (AP) sites are ubiquitous types of damage that must be efficiently repaired to prevent mutations. These damages can occur in both the nuclear and mitochondrial genomes. Base excision repair (BER) is the frontline pathway for identifying and excising damaged DNA bases in both of these cellular compartments. Recent advances demonstrate that BER does not operate as an isolated pathway but rather dynamically interacts with components of other DNA repair pathways to modulate and coordinate BER functions. We define the coordination and interaction between DNA repair pathways as pathway crosstalk. Numerous BER proteins are modified and regulated by post-translational modifications (PTMs), and PTMs could influence pathway crosstalk. Here, we present recent advances on BER/DNA repair pathway crosstalk describing specific examples and also highlight regulation of BER components through PTMs. We have organized and reported functional interactions and documented PTMs for BER proteins into a consolidated summary table. We further propose the concept of DNA repair hubs that coordinate DNA repair pathway crosstalk to identify central protein targets that could play a role in designing future drug targets.

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as 56Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a thresholdlike response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.

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.