Transcription-coupled Nucleotide Excision Repair Research Articles

Targeting transcription-coupled nucleotide excision repair overcomes resistance in chronic lymphocytic leukemia.

Treatment resistance becomes a challenge at some point in the course of most patients with chronic lymphocytic leukemia (CLL). This applies to fludarabine-based regimens, and is also an increasing concern in the era of more targeted therapies. As cells with low-replicative activity rely on repair that triggers checkpoint-independent noncanonical pathways, we reasoned that targeting the nucleotide excision repair (NER) reaction addresses a vulnerability of CLL and might even synergize with fludarabine, which blocks the NER gap-filling step. We interrogated here especially the replication-independent transcription-coupled-NER ((TC)-NER) in prospective trial patients, primary CLL cultures, cell lines and mice. We screen selected (TC)-NER-targeting compounds as experimental (illudins) or clinically approved (trabectedin) drugs. They inflict transcription-stalling DNA lesions requiring TC-NER either for their removal (illudins) or for generation of lethal strand breaks (trabectedin). Genetically defined systems of NER deficiency confirmed their specificity. They selectively and efficiently induced cell death in CLL, irrespective of high-risk cytogenetics, IGHV status or clinical treatment history, including resistance. The substances induced ATM/p53-independent apoptosis and showed marked synergisms with fludarabine. Trabectedin additionally perturbed stromal-cell protection and showed encouraging antileukemic profiles even in aggressive and transforming murine CLL. This proof-of-principle study established (TC)-NER as a mechanism to be further exploited to resensitize CLL cells.

Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine

Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully.In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these findings are due to redundancy in the process, and that other enzymes substitute for the lack of MTH1, however the present study cannot determine whether or not the 2′-deoxyribonucleotide pool is the source of urinary 8-oxodGuo. On the basis of the above, urinary 8-oxodGuo is most accurately defined as a non-invasive biomarker of oxidative stress, derived from oxidatively generated damage to 2′-deoxyguanosine.

FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.

Stabilization of Ultraviolet (UV)-stimulated Scaffold Protein A by Interaction with Ubiquitin-specific Peptidase 7 Is Essential for Transcription-coupled Nucleotide Excision Repair

UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER.

Fanconi anemia pathway regulates convergent transcription-induced cell death at trinucleotide repeats in human cells.

Almost 20 incurable neurodegenerative disorders are caused by trinucleotide repeat (TNR) expansion beyond a certain threshold, with disease time of onset and severity positively correlating with repeat length. Typically, long TNRs display a bias toward further expansion and repeats continue to expand not only during germline transmissions from parents to offspring, but also remain highly unstable in somatic tissues of patients. Hence, understanding TNR instability mechanisms sheds light on underlying disease pathology. Recently, we showed that activated ATR is the major signal for convergent-transcription-induced cell death at CAG repeats and is regulated by the mismatch repair (MMR) pathway. Additionally, components of other DNA repair pathways such as transcription-coupled nucleotide excision repair (TC-NER) and R-loop resolution by RNaseH reduce cell death. Because activated ATR signals the Fanconi anemia (FA) pathway of interstrand crosslink DNA repair, we asked whether the FA pathway also modulates convergent-transcription-induced cell death at expanded CAG repeats. We show here that siRNA knockdown of FA components-FANCI, FANCJ, FANCM, FANCA, and FANCD2-decreases cell death, suggesting that FA proteins, like MMR proteins, are activators of cell death during convergent transcription.

Mismatch repair enhances convergent transcription-induced cell death at trinucleotide repeats by activating ATR.

Trinucleotide repeat (TNR) expansion beyond a certain threshold results in some 20 incurable neurodegenerative disorders where disease anticipation positively correlates with repeat length. Long TNRs typically display a bias toward further expansion during germinal transmission from parents to offspring, and then are highly unstable in somatic tissues of affected individuals. Understanding mechanisms of TNR instability will provide insights into disease pathogenesis. Previously, we showed that enhanced convergent transcription at long CAG repeat tracks induces TNR instability and cell death via ATR activation. Components of TC-NER (transcription-coupled nucleotide excision repair) and RNaseH enzymes that resolve RNA/DNA hybrids oppose cell death, whereas the MSH2 component of MMR (mismatch repair) enhances cell death. The exact role of the MMR pathway during convergent transcription-induced cell death at CAG repeats is not well understood. In this study, we show that siRNA knockdowns of MMR components-MSH2, MSH3, MLHI, PMS2, and PCNA-reduce DNA toxicity. Furthermore, knockdown of MSH2, MLH1, and PMS2 significantly reduces the frequency of ATR foci formation. These observations suggest that MMR proteins activate DNA toxicity by modulating ATR foci formation during convergent transcription.

A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair.

Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein.

Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts.

The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) in cells. We also found that the N(2)-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription, where liquid chromatography-tandem mass spectrometry is employed for the identification, and sometimes quantification, of the progeny products arising from the replication or transcription of lesion-bearing DNA substrates in vitro and in mammalian cells. We also highlight their applications to lesion bypass, mutagenesis, and repair studies of three representative types of DNA lesions, that is, the methylglyoxal-induced N(2)-CEdG, oxidatively induced 8,5'-cyclopurine-2'-deoxynucleosides, and regioisomeric alkylated thymidine lesions. Specially, we discuss the similar and distinct effects of the minor-groove DNA lesions including N(2)-CEdG and O(2)-alkylated thymidine lesions, as well as the major-groove O(4)-alkylated thymidine lesions on DNA replication and transcription machinery. For example, we found that the addition of an alkyl group to the O(4) position of thymine may facilitate its preferential pairing with guanine and thus induce exclusively the misincorporation of guanine nucleotide opposite the lesion, whereas alkylation of thymine at the O(2) position may render the nucleobase unfavorable in pairing with any of the canonical nucleobases and thus exhibit promiscuous miscoding properties during DNA replication and transcription. The MS-based strategies described herein should be generally applicable for quantitative measurement of the biological consequences and repair of other DNA lesions in vitro and in cells.

The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair

Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.

Unique features of trabectedin mechanism of action.

Trabectedin (Yondelis®, ET-743) is a marine-derived natural product that was initially isolated from the marine ascidian Ecteinascidia turbinata and is currently prepared synthetically. Trabectedin is used as a single agent for the treatment of patients with soft tissue sarcoma after failure of doxorubicin or ifosfamide or who are unsuited to receive these agents, and in patients with relapsed, platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin. Trabectedin presents a complex mechanism of action affecting key cell biology processes in tumor cells as well as in the tumor microenvironment. The inhibition of trans-activated transcription and the interaction with DNA repair proteins appear as a hallmark of the antiproliferative activity of trabectedin. Inhibition of active transcription is achieved by an initial direct mechanism that involves interaction with RNA polymerase II, thereby inducing its ubiquitination and degradation by the proteasome. This subsequently modulates the production of cytokines and chemokines by tumor and tumor-associated macrophages. Another interesting effect on activated transcription is mediated by the displacement of oncogenic transcription factors from their target promoters, thereby affecting oncogenic signaling addiction. In addition, it is well established that DNA repair systems including transcription-coupled nucleotide excision repair and homologous recombination play a role in the antitumor activity of trabectedin. Ongoing studies are currently addressing how to exploit these unique mechanistic features of trabectedin to combine this agent either with immunological or microenvironmental modulators or with classical chemotherapeutic agents in a more rational manner.

Abstract C55: Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer

Abstract Platinum-based therapies, both cisplatin and carboplatin, are utilized as part of first-line standard of care regimens for advanced non-small cell lung cancer (NSCLC), but overall response rate and overall survival remain limited, with a 5-year survival rate of 18%. Therefore, agents that can improve responses and survival are needed. Pevonedistat (MLN4924) is an investigational small molecule inhibitor of the Nedd8-activating enzyme (NAE) currently in Phase Ib clinical trials. Nedd8 is a small ubiquitin-like protein whose activation of the cullin family of E3 ubiquitin ligases is dependent on NAE activity. Importantly, pevonedistat synergized with carboplatin in a cell viability assay for 6 of 20 NSCLC cell lines tested in vitro, and synergy was also detected in the triple combination of carboplatin, pevonedistat, and paclitaxel. Order of addition experiments in the A549 NSCLC cell line demonstrated a benefit of simultaneous treatment or addition of pevonedistat prior to platinum treatment, while a reduced combination benefit was observed when cisplatin was added 24-48 hrs before pevonedistat. Levels of NAE pathway inhibition were similar in pevonedistat and pevonedistat+cisplatin treated cells, indicating that a drug-drug interaction was not likely the cause of the synergy. Using patient-derived xenograft (PDX) models a combination benefit of carboplatin and pevonedistat was demonstrated in a carboplatin-insensitive model but a similar improvement in response was not observed in a carboplatin-sensitive model, adding to evidence that pevonedistat might target platinum resistance mechanisms. To evaluate the mechanism of synergy between pevonedistat and platinum, in vitro experiments with RNAi were performed in 4 cell lines. These studies identified 2 mechanisms of synergy between pevonedistat and platinums in vitro. Depletion of genes within the TC-NER (transcription-coupled nucleotide excision repair) and ICR (interstrand crosslink repair) pathways reduced the synergy between pevonedistat and platinum, with the contribution of each pathway varying by cell line. Because these pathways are also implicated in the response to single agent platinums, the results suggest pevonedistat could delay completion of platinum-induced DNA repair and therefore result in enhanced cell death. Inhibition of neddylation of the E3 ubiquitin ligase CUL4-RBX1-DDB1-ERCC8 by pevonedistat may delay the TC-NER pathway, providing a direct mechanism of resistance reversal by pevonedistat. The combination of pevonedistat with carboplatin and paclitaxel is under evaluation as part of a phase 1b trial in adult patients with solid tumors (NCT01862328). Citation Format: David C. Bouck, Khristofer Garcia, Jonathan L. Blank, Xiaozhen J. Liu, Hugues Bernard, Allison J. Berger, Mike Kuranda, Erik Koenig, Eric S. Lightcap. Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C55.

Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26.

Transcription coupled nucleotide excision repair (TC-NER) is believed to be triggered by an RNA polymerase stalled at a lesion in the transcribed strand of actively transcribed genes. Rad26, a DNA-dependent ATPase in the family of SWI2/SNF2 chromatin remodeling proteins, plays an important role in TC-NER in Saccharomyces cerevisiae. However, Rad26 is not solely responsible for TC-NER and Rpb9, a nonessential subunit of RNA polymerase II (RNAP II), is largely responsible for Rad26-independent TC-NER. The Rad26-dependent and Rpb9-dependent TC-NER have different efficiencies in genes with different transcription levels and in different regions of a gene. Rad26 becomes entirely or partially dispensable for TC-NER in the absence of Rpb4, another nonessential subunit of RNAP II, or a number of transcription elongation factors (Spt4, Spt5 and the RNAP II associated factor complex). Rad26 may not be a true transcription-repair coupling factor that recruits the repair machinery to the damaged sites where RNAP II stalls. Rather, Rad26 may facilitate TC-NER indirectly, by antagonizing the action of TC-NER repressors that normally promote transcription elongation. The underlying mechanism of how Rad26 functions in TC-NER remains to be elucidated.

DNA damage mediated transcription arrest: Step back to go forward.

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored.

Abstract 2550: FANCA protein is involved in the homologous recombination repair of sapacitabine-induced DNA damage

Abstract Fanconi anemia (FA) is an autosomal disorder characterized by developmental abnormalities, early-onset bone marrow failure, and a predisposition to cancer. FA cells are defective in DNA repair, and exhibit hypersensitivity to DNA interstrand cross-links (ICLs) and a high degree of chromosomal aberrations. A majority of the FA proteins have been shown to act as signal transducers and scaffolding proteins to employ other pathways to repair DNA. Specifically the FA group A (FANCA) protein has been indicated as an integral component in the early step of homologous recombination (HR) of double strand breaks (DSB). Sapacitabine, the orally bioavailable prodrug of the nucleoside analog CNDAC, has presented favorable clinical activity in advanced acute leukemias and myelodysplastic syndromes. CNDAC kills cells by a unique mechanism of action. Incorporation of CNDAC triphosphate into DNA results in a beta elimination reaction that causes single-strand break (SSB) that is terminated by the analogue. DNA replication over this nick converts the nick to a one-ended double-strand break (DSB) upon collapse of the DNA replication fork. The CNDAC-induced DSBs, which are potentially lethal, are repaired predominantly through homologous recombination (HR), relative to other repair pathways, such as base excision repair, transcription-coupled nucleotide excision repair and non-homologous end-joining. CNDAC greatly sensitizes cells lacking HR components, such as ATM, RAD51D, XRCC3 and BRCA2. Based on the evidence that FANCA associates with proteins involved with HR, we hypothesize that loss of FANCA may also sensitize cells to CNDAC. FANCA-deficient cells from an individual with Fanconi anemia as well FANCA repleted cells were utilized to investigate the role of the FANCA protein in the repair of damage caused by CNDAC. Cells were treated with CNDAC or mitomycin C (MMC), a crosslinking agent. We found that FANCA deficient cells were sensitized to CNDAC about 5-fold as compared with FANCA repleted cells, while they were sensitized by 6-fold when treated with MMC. Cells were treated with 1 μM CNDAC for either 24 or 48 hr before chromosome spread samples were collected to examine the genome integrity. Chromosome aberrations were increased in response to CNDAC treatment, and cells lacking FANCA protein exhibited more chromosomal abnormalities compared with FANCA repleted cells. Rad51 foci, diagnostic indicators of active HR, were present in both lines after 24 or 48 hr of CNDAC treatment; however, FANCA deficient cells displayed attenuated accumulation of Rad51 foci in response to CNDAC as compared with the FANCA repleted cells. These findings indicate that FANCA is involved in the HR repair of CNDAC-induced DNA lesions. Because FANCA is one component of the core complex of the Fanconi repair pathway, other proteins in this group may confer similar sensitization. Citation Format: Yingjun Jiang, Xiaojun Liu, William Plunkett. FANCA protein is involved in the homologous recombination repair of sapacitabine-induced DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2550. doi:10.1158/1538-7445.AM2015-2550

The Cockayne Syndrome Natural History (CoSyNH) study: clinical findings in 102 individuals and recommendations for care.

Purpose:Cockayne syndrome (CS) is a rare, autosomal-recessive disorder characterized by microcephaly, impaired postnatal growth, and premature pathological aging. It has historically been considered a DNA repair disorder; fibroblasts from classic patients often exhibit impaired transcription-coupled nucleotide excision repair. Previous studies have largely been restricted to case reports and small series, and no guidelines for care have been established.Genet Med 18 5, 483–493.Methods:One hundred two study participants were identified through a network of collaborating clinicians and the Amy and Friends CS support groups. Families with a diagnosis of CS could also self-recruit. Comprehensive clinical information for analysis was obtained directly from families and their clinicians.Genet Med 18 5, 483–493.Results and Conclusion:We present the most complete evaluation of Cockayne syndrome to date, including detailed information on the prevalence and onset of clinical features, achievement of neurodevelopmental milestones, and patient management. We confirm that the most valuable prognostic factor in CS is the presence of early cataracts. Using this evidence, we have created simple guidelines for the care of individuals with CS. We aim to assist clinicians in the recognition, diagnosis, and management of this condition and to enable families to understand what problems they may encounter as CS progresses.Genet Med 18 5, 483–493.

Large expansion of CTG•CAG repeats is exacerbated by MutSβ in human cells.

Trinucleotide repeat expansion disorders (TRED) are caused by genomic expansions of trinucleotide repeats, such as CTG and CAG. These expanded repeats are unstable in germline and somatic cells, with potential consequences for disease severity. Previous studies have demonstrated the involvement of DNA repair proteins in repeat instability, although the key factors affecting large repeat expansion and contraction are unclear. Here we investigated these factors in a human cell model harboring 800 CTG•CAG repeats by individually knocking down various DNA repair proteins using short interfering RNA. Knockdown of MSH2 and MSH3, which form the MutSβ heterodimer and function in mismatch repair, suppressed large repeat expansions, whereas knockdown of MSH6, which forms the MutSα heterodimer with MSH2, promoted large expansions exceeding 200 repeats by compensatory increases in MSH3 and the MutSβ complex. Knockdown of topoisomerase 1 (TOP1) and TDP1, which are involved in single-strand break repair, enhanced large repeat contractions. Furthermore, knockdown of senataxin, an RNA/DNA helicase which affects DNA:RNA hybrid formation and transcription-coupled nucleotide excision repair, exacerbated repeat instability in both directions. These results indicate that DNA repair factors, such as MutSβ play important roles in large repeat expansion and contraction, and can be an excellent therapeutic target for TRED.

601. Gene Therapy for Cockayne Syndrome

Cockayne Syndrome (CS) is a rare autosomal recessive genetic disorder. The majority of CS cases involve mutations in either the CSA (25%) or CSB (75%) gene, leading to defective transcription-coupled nucleotide excision repair and RNA polymerase II mediated transcription. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, causing mental retardation, post-natal growth defects, pigmented retinopathy, cataracts, dermal UV sensitivity, organ dysfunction and shortened life expectancy.CSA and CSB single mutant mice display retinal degeneration with age and ocular sensitivity to ionizing radiation. Because limited publications on these mouse models exist we began by characterizing the retinal dysfunction through histology and electroretinography (ERG). We found that the retinal degeneration in CSA and CSB mutant mice (C57Bl6 background) was occurring over a slower time course (over 1 year) than previously reported. Therefore we aimed to increase the rate of retinal degeneration in these animals using UV light exposure, whole body radiation, or high intensity light exposure. Our goal was to increase the speed of retinal degeneration to a timeframe that is more readily studied for the purposes of gene therapy correction. Preliminary data indicates that we can cause a decrease in the scotopic ERG (b-wave) 2 weeks after high-dose ultraviolet light exposure (100000J/m^2), a change not seen in C57Bl6 mice at the same UV dose. There was also a decrease in b-wave amplitude following radiation exposure (2-3 gray) in the CSB mouse model that was not seen in C57Bl6 mice. Histology on these mice is currently pending. Improving the rate of photoreceptor loss will allow us to perform rAAV based gene therapy to deliver the CSB gene back to the retina and ascertain whether UV and radiation resistance can be restored.While single mutant CSA and CSB mice do not have significant neurological deficits, when crossed with XPA deficient mice they develop clinical signs similar to severely affected human patients. Signs in these mice include neurologic deficits, poor weight gain and death around postnatal day 21. These mice could be ideal candidates to show that correction of both the central nervous system disease as well as the retinal degeneration is possible in a single model. To determine if both the brain and retina could be targeted in the same animal (and therefore model what may be necessary in the human patients) we have looked at targeting both organs simultaneously using either a single IV injection in neonatal mice or a combined intra-cerebral ventricular (ICV) injection and IV injection in the same animal. We found that delivery of the rAAV vector (regardless of serotype tested) into the retro-orbital sinus at postnatal day 1-3 leads to widespread transduction of the retina. Temporal vein delivery postnatal day 1-3 only resulted in transduction of the inner layer of the retina, not the photoreceptors. When ICV and IV injections were combined in neonates widespread brain and retinal transduction was achieved.These preliminary data indicate that a murine model for retinal gene therapy of CS is feasible and that potentially both the retina and brain could be targeted in a single model.

Polymorphisms in rs 13181 of ERCC2 gene in the personnel of Stepnogorsk gorno chemiches kombinat, Kazakhstan

The possibility of mutations in human somatic cells induced by action of radiation in low doses is a subject of investigations and discussion. The protein encoded by ERCC2 (XPD) gene is involved in transcription‐coupled nucleotide excision repair and is associated with some types of cancer. The aim of this study was to determine the influence of chronic action of radiation in low doses to the level of the single nucleotide polymorphisms in DNA of the personnel of uranium enrichment plant, Akmola, Kazakhstan. The sample set consisted of 234 (52–Kazakhs, 182–Russians) men workers of and the control group includes 289 (129–Kazakhs, 160–Russians). Genotyping was performed by restriction fragments length polymorphism analysis of PCR products of the rs13181 site (T/G) of (T/G) of ERCC2 gene using PstI. In the case‐control studythe allele frequencies and genotypes distribution were compared in the groups of personnel and controlsusing STATISTICA v. 5.0 software.The differences in allele frequencies and genotypes distribution in the groups of personnel and controls were evaluated neither in Kazakh nor in Russian groups. At the next stage comparing of allele frequencies and genotypes distribution was performed in the subgroups of the workers divided according accumulated dose: less than 40 mZv, 40‐100 mZv.The differences were found in genotype distribution only in the group of 50 Russian workers with accumulated dose 40–100 mZv compared with 100 of controls: χ2=6.796, p=0.033, OR=1.363, 95%CI=0.844‐2.201. The group of persons who accumulated dose more than 100 mZv encountered only 5 persons and was not included in calculations.

DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes.

The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5′-3′ direction with slow recovery and TC-NER at the 3′ end of long genes. RNA synthesis resumed fully at the 3′-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.

Longevity through DNA damage tolerance

Longevity through DNA damage tolerance