Bruce Demple

DNA repair in mammalian mitochondria: Much more than we thought?

For many years, the repair of most damage in mitochondrial DNA (mtDNA) was thought limited to short‐patch base excision repair (SP‐BER), which replaces a single nucleotide by the sequential action of DNA glycosylases, an apurinic/apyrimidinic (AP) endonuclease, the mitochondrial DNA polymerase γ, an abasic lyase activity, and mitochondrial DNA ligase. However, the likely array of lesions inflicted on mtDNA by oxygen radicals and the possibility of replication errors and disruptions indicated that such a restricted repair repertoire would be inadequate. Recent studies have considerably expanded our knowledge of mtDNA repair to include long‐patch base excision repair (LP‐BER), mismatch repair, and homologous recombination and nonhomologous end‐joining. In addition, elimination of mutagenic 8‐oxodeoxyguanosine triphosphate (8‐oxodGTP) helps prevent cell death due to the accumulation of this oxidation product in mtDNA. Although it was suspected for many years that irreparably damaged mtDNA might be targeted for degradation, only recently was clear evidence provided for this hypothesis. Therefore, multiple DNA repair pathways and controlled degradation of mtDNA function together to maintain the integrity of mitochondrial genome. Environ. Mol. Mutagen., 2010.

Essential role for mammalian apurinic/apyrimidinic (AP) endonuclease Ape1/Ref-1 in telomere maintenance

Significance Base excision repair (BER) is the predominant system correcting simple DNA base lesions formed by oxidation or other DNA-damaging agents. Repair of apurinic/apyrimidinic (AP) sites arising in the genome spontaneously or as intermediates of BER is critical owing to their toxic and mutagenic effects. Ape1/Ref-1 is the major AP endonuclease that initiates the processing of AP sites, allowing normal transcription and DNA synthesis to resume. In this study, we report a key role for Ape1/Ref-1 in telomere maintenance. Our findings suggest a direct link between BER and telomere dynamics, highlighting the potential contribution of oxidative DNA damage repair activities on telomere dysfunction in cancer, premature aging, or autoimmune diseases. The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for Ape1-dependent DNA repair activity, deeper molecular mechanisms of the fundamental role of Ape1 in cell survival have not been defined. Here, we report that Ape1 is an essential factor stabilizing telomeric DNA, and its deficiency is associated with telomere dysfunction and segregation defects in immortalized cells maintaining telomeres by either the alternative lengthening of telomeres pathway (U2OS) or telomerase expression (BJ-hTERT), or in normal human fibroblasts (IMR90). Through the expression of Ape1 derivatives with site-specific changes, we found that the DNA repair and N-terminal acetylation domains are required for the Ape1 function at telomeres. Ape1 associates with telomere proteins in U2OS cells, and Ape1 depletion causes dissociation of TRF2 protein from telomeres. Consistent with this effect, we also observed that Ape1 depletion caused telomere shortening in both BJ-hTERT and in HeLa cells. Thus, our study describes a unique and unpredicted role for Ape1 in telomere protection, providing a direct link between base excision DNA repair activities and telomere metabolism.

Roles of Rev1, Pol ζ, Pol32 and Pol η in the bypass of chromosomal abasic sites in Saccharomyces cerevisiae

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3Delta and rev7Delta) or enhanced by (rev1Delta) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression.

Enzyme mechanism-based, oxidative DNA–protein cross-links formed with DNA polymerase β in vivo

Significance Oxidative DNA damage is a by-product of aerobic metabolism and various genotoxic agents, including many cancer therapy regimens. Important DNA lesions include 2-deoxyribonolactone (dL), a major type of oxidized abasic (AP) site. In addition to the cytotoxic and mutagenic effects of non-oxidized AP sites, dL poses another threat: in vitro, dL traps some DNA repair enzymes in covalent DNA–protein cross-links (DPC) via their AP lyase activity. A prime example is DNA polymerase β (Polβ), which is critical for base excision DNA repair. Here we show that oxidative Polβ-DPC form robustly during attempted DNA repair in mammalian cells treated with dL-inducing oxidants. Mammalian cells remove Polβ-DPC surprisingly rapidly by a mechanism dependent on proteasome activity. Their accumulation is toxic. Free radical attack on the C1′ position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA–protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-DPC under 1% O2 than under 21% O2) and even more robustly with the “chemical nuclease” 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-DPC had an unexpectedly short half-life (∼30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them.

A DNA-Based Nanomechanical Device Used To Characterize the Distortion of DNA by Apo-SoxR Protein

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple-crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In our case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to ~2 bp. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding of the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

Risky repair: DNA-protein crosslinks formed by mitochondrial base excision DNA repair enzymes acting on free radical lesions ☆

Oxygen is both necessary and dangerous for aerobic cell function. ATP is most efficiently made by the electron transport chain, which requires oxygen as an electron acceptor. However, the presence of oxygen, and to some extent the respiratory chain itself, poses a danger to cellular components. Mitochondria, the sites of oxidative phosphorylation, have defense and repair pathways to cope with oxidative damage. For mitochondrial DNA, an essential pathway is base excision repair, which acts on a variety of small lesions. There are instances, however, in which attempted DNA repair results in more damage, such as the formation of a DNA-protein crosslink trapping the repair enzyme on the DNA. That is the case for mitochondrial DNA polymerase γ acting on abasic sites oxidized at the 1-carbon of 2-deoxyribose. Such DNA-protein crosslinks presumably must be removed in order to restore function. In nuclear DNA, ubiquitylation of the crosslinked protein and digestion by the proteasome are essential first processing steps. How and whether such mechanisms operate on DNA-protein crosslinks in mitochondria remains to be seen.

Ape1 guides DNA repair pathway choice that is associated with drug tolerance in glioblastoma

Ape1 is the major apurinic/apyrimidinic (AP) endonuclease activity in mammalian cells, and a key factor in base-excision repair of DNA. High expression or aberrant subcellular distribution of Ape1 has been detected in many cancer types, correlated with drug response, tumor prognosis, or patient survival. Here we present evidence that Ape1 facilitates BRCA1-mediated homologous recombination repair (HR), while counteracting error-prone non-homologous end joining of DNA double-strand breaks. Furthermore, Ape1, coordinated with checkpoint kinase Chk2, regulates drug response of glioblastoma cells. Suppression of Ape1/Chk2 signaling in glioblastoma cells facilitates alternative means of damage site recruitment of HR proteins as part of a genomic defense system. Through targeting “HR-addicted” temozolomide-resistant glioblastoma cells via a chemical inhibitor of Rad51, we demonstrated that targeting HR is a promising strategy for glioblastoma therapy. Our study uncovers a critical role for Ape1 in DNA repair pathway choice, and provides a mechanistic understanding of DNA repair-supported chemoresistance in glioblastoma cells.

Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro-Inflammation in Brain, Cognitive Deficits, and Alterations in Apurinic Endonuclease 1

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

How are base excision DNA repair pathways deployed in vivo

Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.

Abstract B26: Ape1/Ref-1 directs DNA repair pathway choice, linked to Chk2 signaling, critical for glioblastoma response to chemotherapy

The predominant human apurinic/apyrimidinic (AP) endonuclease 1, Ape1 (also known as Ref-1) plays a central role in the pathways of base excision repair (BER) of DNA. BER is the major system for correcting the oxidative DNA damage that arises endogenously as a result of metabolism. Spontaneous hydrolytic decay of DNA or DNA glycosylase-mediated hydrolysis of N-glycosyl bonds generates AP sites at an estimated frequency of >10,000 per day in each human cell. If unrepaired, non-instructional AP sites potently block DNA replication and thereby cause toxicity. Thus, the proper repair of AP sites is crucial for maintaining genome stability. Genetic studies in mice support the essential role of Ape1 in cellular viability. In this study, we have explored the molecular details of Ape1-mediated genome maintenance mechanisms, and applied glioblastoma multiforme (GBM) tumor model for investigation of the functional significance of the Ape1-mediated cell responses to chemotherapy. Here we present evidence that Ape1 links to a Chk2-associated tumor suppressor pathway, and is necessary for proper execution of DNA damage-induced checkpoints. Furthermore, our study revealed an unexpected role for Ape1 in BRCA1-mediated homologous recombination (HR) repair of DNA breaks, which is crucial for directing DNA repair pathway choices. The analyses of the TCGA data set for GBM showed that Ape1 and Chk2 are coordinately regulated in glioblastoma multiforme, and increased expression of APEX1 gene coding for Ape1 positively correlates with better survival of glioblastoma patients. GBM is the most common and deadly type of primary brain tumor. The current therapy remains relatively ineffective due to diffuse infiltration and intrinsic resistance of GBM cells. Thus, there is an urgent need for rational treatment protocols for the GBM cure. Our study provides evidence for critical importance of the survival network of Ape1 and Chk2 in GBM response to chemotherapy. Regulation of the equilibrium between Ape1 and Chk2 enables tumor cells switch between DNA repair pathways to favor cell survival. Identification of tumor-specific pre-dominant DNA repair pathways and interruption of such survival networks can be a useful strategy for designing novel therapies for GBM. Citation Format: Thomas Stroebel, Sibylle Madlener, Serkan Tuna, Sarah Vose, Daniella Morse, Bakhos A. Tannous, Tonny Lagerweij, Thomas Wurdinger, Christine Marosi, Irene Slavc, Klemens Vierlinger, Okay Saydam, Brendan D. Price, Bruce Demple, Nurten Saydam. Ape1/Ref-1 directs DNA repair pathway choice, linked to Chk2 signaling, critical for glioblastoma response to chemotherapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B26.