Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Functional DNA repair capacity assays : a focus on base excision repair

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.The integrity of our DNA is challenged by roughly 100,000 lesions per cell on a daily basis. Failure to cope with DNA damage can lead to cancer, immunodeficiency and degenerative disease. Quantitating and understanding an individual's DNA repair capacity may enable us to predict and prevent disease in a personalized manner. Base Excision Repair (BER) is known for the recognition and repair of endogenous and exogenous mutagenic non-helix-distorting lesions produced by DNA base alkylation, deamination and oxidation. BER is initiated by the action of one of eleven DNA glycosylases known-to-date. Many studies have shown that levels of these glycosylases can vary between individuals, suggesting a basis for inter-individual differences in DNA repair capacity. Moreover, the methods for measuring DNA repair capacity used so far are cumbersome, time consuming, low throughput and only allow for the analysis of one glycosylase at a time. We have taken a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several DNA glycosylases and their immediate downstream endonuclease (APE1) can be tested simultaneously, at single-cell resolution, under physiological conditions. Taking advantage of the transcriptional properties of several DNA lesions we have designed and engineered specific fluorescent reporter plasmids for OGG1, AAG, MUTYH, UNG and APE1. Inter-individual differences in DNA repair capacity of a panel of cell lines derived from healthy individuals have been measured. Regression models that incorporate these measurements have been developed in order to predict cellular sensitivity to the chemotherapeutic and DNA damaging agents 5-FU, H₂O₂ and MMS, with the interest of understanding the contributions that these differences can have on personalized disease prevention and treatment. Finally, we have conducted a pilot population study with 56 healthy subjects where we implemented all the methods developed in order to determine the feasibility of measuring DNA repair capacity variations in a healthy human population. Additionally, we report the discovery of a novel in vivo role of the TC-NER pathway in the repair of the lipid-peroxidation product, 3,N⁴-etheno-cytosine.by Isaac Alexander Chaim.Ph. D

Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms

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. Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER) pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg). Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS) undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose) polymerase 1 (Parp1). Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN) sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD⁺ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.National Institutes of Health (U.S.) (Grant R01- ES022872)Ellison Medical Foundation (Award AG-SS-3046-12

In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual diff erences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H₂O₂, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals. Keywords:DNA repair; transcriptional mutagenesis; DNA glycosylase; apurinic/apyrimidinic; endonuclease 1; base excision repairNational Institutes of Health (U.S.) (Award DPI-ES022576)National Institutes of Health (U.S.) (Grant R01-CA075576)National Institutes of Health (U.S.) (Grant R01-CA55042)National Institutes of Health (U.S.) (Grant R01-CA149261)National Institutes of Health (U.S.) (Grant R01-ES022872)National Institutes of Health (U.S.) (Grant P30-ES02109

A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3, N[superscript4]-ethenocytosine

Etheno (ε) DNA base adducts are highly mutagenic lesions produced endogenously via reactions with lipid peroxidation (LPO) products. Cancer-promoting conditions, such as inflammation, can induce persistent oxidative stress and increased LPO, resulting in the accumulation of ε-adducts in different tissues. Using a recently described fluorescence multiplexed host cell reactivation assay, we show that a plasmid reporter bearing a site-specific 3,N4-ethenocytosine (εC) causes transcriptional blockage. Notably, this blockage is exacerbated in Cockayne Syndrome and xeroderma pigmentosum patient-derived lymphoblastoid and fibroblast cells. Parallel RNA-Seq expression analysis of the plasmid reporter identifies novel transcriptional mutagenesis properties of εC. Our studies reveal that beyond the known pathways, such as base excision repair, the process of transcription-coupled nucleotide excision repair plays a role in the removal of εC from the genome, and thus in the protection of cells and tissues from collateral damage induced by inflammatory responses.National Institutes of Health (U.S.) (Directors Pioneer Award [DPI-ES022576])National Institutes of Health (U.S.) (R01-CA075576)National Institutes of Health (U.S.) (R01-CA55042)National Institutes of Health (U.S.) (R01-CA149261)National Institutes of Health (U.S.) (P30-ES02109)National Institutes of Health (U.S.). Intramural Research ProgramNational Institute on AgingLDS discretionary fund

mAagTg CGN sensitivity to MMS is not dependent on NAD+, pyruvate, caspases, Rip1K, calcium fluxes, or MPT.

<p>(A) NAD⁺ pre-treatment did not rescue <i>mAagTg</i> neuron sensitivity to MMS. Errors bars denote standard deviation from the mean. <i>mAagTg</i> n = 3. (B) Pyruvate did not rescue <i>mAagTg</i> neuron sensitivity to MMS. Errors bars denote standard deviation from the mean. <i>mAagTg</i> n = 1. *** p<0.001 using Student’s standard two-tailed T-test comparing MMS and MMS + Veliparib. (C) Primary <i>mAagTg</i> CGN sensitivity to MMS is not dependent on caspase activation, Rip1 kinase activity, calcium fluxes, or mitochondrial permeability transition (MPT). Inhibitors used include zVad-fmk (zVad), n = 6, Necrostatin-1 (Nec-1), n = 3, BAPTA-AM, n = 3, and cyclosporin A (CsA), n = 3. Errors bars denote standard error from the mean. *** p<0.001 using Student’s standard two-tailed T-test comparing MMS to MMS+Veliparib.</p