Elise Fouquerel

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

Nicotinamide adenine dinucleotide, NAD(+), is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD(+) and NADH). NAD(+) is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD(+) as a substrate. In this review, we highlight the significance of NAD(+) catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.

Oxidative guanine base damage regulates human telomerase activity

Changes in telomere length are associated with degenerative diseases and cancer. Oxidative stress and DNA damage have been linked to both positive and negative alterations in telomere length and integrity. Here we examined how the common oxidative lesion 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG) regulates telomere elongation by human telomerase. When 8-oxoG is present in the dNTP pool as 8-oxodGTP, telomerase utilization of the oxidized nucleotide during telomere extension is mutagenic and terminates further elongation. Depletion of MTH1, the enzyme that removes oxidized dNTPs, increases telomere dysfunction and cell death in telomerase-positive cancer cells with shortened telomeres. In contrast, a preexisting 8-oxoG within the telomeric DNA sequence promotes telomerase activity by destabilizing the G-quadruplex DNA structure. We show that the mechanism by which 8-oxoG arises in telomeres, either by insertion of oxidized nucleotides or by direct reaction with free radicals, dictates whether telomerase is inhibited or stimulated and thereby mediates the biological outcome.

Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts

Ultraviolet light induces cyclobutane pyrimidine dimers (CPD) and pyrimidine(6–4)pyrimidone photoproducts, which interfere with DNA replication and transcription. Nucleotide excision repair (NER) removes these photoproducts, but whether NER functions at telomeres is unresolved. Here we use immunospot blotting to examine the efficiency of photoproduct formation and removal at telomeres purified from UVC irradiated cells at various recovery times. Telomeres exhibit approximately twofold fewer photoproducts compared with the bulk genome in cells, and telomere-binding protein TRF1 significantly reduces photoproduct formation in telomeric fragments in vitro. CPD removal from telomeres occurs 1.5-fold faster than the bulk genome, and is completed by 48 h. 6–4PP removal is rapidly completed by 6 h in both telomeres and the overall genome. A requirement for XPA protein indicates the mechanism of telomeric photoproduct removal is NER. These data provide new evidence that telomeres are partially protected from ultraviolet irradiation and that NER preserves telomere integrity.

Synthesis and Characterization of DNA Minor Groove Binding Alkylating Agents

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

DNA damage processing at telomeres: The ends justify the means

Telomeres at chromosome ends are nucleoprotein structures consisting of tandem TTAGGG repeats and a complex of proteins termed shelterin. DNA damage and repair at telomeres is uniquely influenced by the ability of telomeric DNA to form alternate structures including loops and G-quadruplexes, coupled with the ability of shelterin proteins to interact with and regulate enzymes in every known DNA repair pathway. The role of shelterin proteins in preventing telomeric ends from being falsely recognized and processed as DNA double strand breaks is well established. Here we focus instead on recent developments in understanding the roles of shelterin proteins and telomeric DNA sequence and structure in processing genuine damage at telomeres induced by endogenous and exogenous DNA damage agents. We will highlight advances in double strand break repair, base excision repair and nucleotide excision repair at telomeres, and will discuss important questions remaining in the field.

The impact of oxidative DNA damage and stress on telomere homeostasis

Telomeres are dynamic nucleoprotein-DNA structures that cap and protect linear chromosome ends. Because telomeres shorten progressively with each replication, they impose a functional limit on the number of times a cell can divide. Critically short telomeres trigger cellular senescence in normal cells, or genomic instability in pre-malignant cells, which contribute to numerous degenerative and aging-related diseases including cancer. Therefore, a detailed understanding of the mechanisms of telomere loss and preservation is important for human health. Numerous studies have shown that oxidative stress is associated with accelerated telomere shortening and dysfunction. Oxidative stress caused by inflammation, intrinsic cell factors or environmental exposures, contributes to the pathogenesis of many degenerative diseases and cancer. Here we review the studies demonstrating associations between oxidative stress and accelerated telomere attrition in human tissue, mice and cell culture, and discuss possible mechanisms and cellular pathways that protect telomeres from oxidative damage.

γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.

Abstract 4: Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma

PARP1 is a key enzyme of the Base Excision Repair (BER) pathway, facilitating the repair of base damage and single-strand DNA breaks. Activated PARP1 synthesizes poly (ADP-ribose) (PAR), triggering chromatin de-condensation to facilitate recruitment of BER proteins to complete repair. PARP1 activation is attenuated upon successful repair of the DNA lesion. However, unrepaired DNA breaks lead to continuous PARP1 activation and cell death. The molecular mechanism underlying PARP1 activation induced cell death was recently revealed as independent from NAD+ depletion. We have shown that PARP1 activation and PAR synthesis affect glycolysis by directly inhibiting the glycolytic enzyme, hexokinase 1 (HK1). Following on these discoveries, we decided to investigate global metabolic changes triggered by hyperactivation of PARP1. For this study, we used gas chromatography mass spectrometry (GC-MS) to quantify over 150 cellular metabolites and Multiple-Reaction Monitoring Liquid Chromatography Mass Spectrometry (MRM LC-MS) to measure NAD+ metabolites. As a model, we tested glioblastoma cells overexpressing methylpurine DNA glycosylase (MPG) to enhance the PARP1-activation response to DNA damage induced by the alkylating agent MNNG. Simultaneously, to monitor independence from the DNA damaging effect of NAD+ depletion, we utilized an inhibitor of NAD+ biosynthesis, FK866. We found that PARP1 activation leads to a strong accumulation of glucose, likely as a secondary effect of HK1 inhibition. In addition, we observed a significant change in the level of other metabolites including an increase in inosine, inosine monophosphate (IMP), cytidine and uridine levels upon PARP1 activation, suggesting an indirect effect of PARP1 activation on purine and pyrimidine metabolism. Ongoing studies will use these global approaches to unravel the complete metabolic response of cancer cells to genotoxic treatment. Citation Format: Anna M. Wilk, Elise Fouquerel, Bobbie Johnston, Samuel A.J. Trammell, Lindsay Schambeau, Joel F. Andrews, Lewis Pannell, Sara J. Cooper, Charles Brenner, Robert W. Sobol. Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4.

Convergence of The Nobel Fields of Telomere Biology and DNA Repair

The fields of telomere biology and DNA repair have enjoyed a great deal of cross‐fertilization and convergence in recent years. Telomeres function at chromosome ends to prevent them from being falsely recognized as chromosome breaks by the DNA damage response and repair machineries. Conversely, both canonical and nonconical functions of numerous DNA repair proteins have been found to be critical for preserving telomere structure and function. In 2009, Elizabeth Blackburn, Carol Greider and Jack Szostak were awarded the Nobel prize in Physiology or Medicine for the discovery of telomeres and telomerase. Four years later, pioneers in the field of DNA repair, Aziz Sancar, Tomas Lindahl and Paul Modrich were recognized for their seminal contributions by being awarded the Nobel Prize in Chemistry. This review is part of a special issue meant to celebrate this amazing achievement, and will focus in particular on the convergence of nucleotide excision repair and telomere biology, and will discuss the profound implications for human health.

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.