Comet assay to measure DNA repair: approach and applications

Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle and occupation, in addition to clinical investigations

ERCC1–XPF targeting to psoralen–DNA crosslinks depends on XPA and FANCD2

The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1–XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1–XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1–XPF recruitment to ICLs is less well u

Detection of oxaliplatin- and cisplatin-DNA lesions requires different global genome repair mechanisms that affect their clinical efficacy

The therapeutic efficacy of cisplatin and oxaliplatin depends on the balance between the DNA damage induction and the DNA damage response of tumor cells. Based on clinical evidence, oxaliplatin is administered to cisplatin-unresponsive cancers, but the underlying molecular causes for this tumor specificity are not clear. Hence, stratification of patients based on DNA repair profiling is not sufficiently utilized for treatment selection. Using a combination of genetic, transcriptomics and imaging approaches, we identified factors that promote global genome nucleotide excision repair (GG-NER) of DNA-platinum adducts induced by oxaliplatin, but not by cisplatin. We show that oxaliplatin-DNA lesions are a poor substrate for GG-NER initiating factor XPC and that DDB2 and HMGA2 are required for efficient binding of XPC to oxaliplatin lesions and subsequent GG-NER initiation. Loss of DDB2 and HMGA2 therefore leads to hypersensitivity to oxaliplatin but not to cisplatin. As a result, low DDB2 levels in different colon cancer cells are associated with GG-NER deficiency and oxaliplatin hypersensitivity. Finally, we show that colon cancer patients with low DDB2 levels have a better prognosis after oxaliplatin treatment than patients with high DDB2 expression. We therefore propose that DDB2 is a promising predictive marker of oxaliplatin treatment efficiency in colon cancer.</p

Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage

Sensitivity and resistance of cells to platinum drug chemotherapy are to a large extent determined by activity of the DNA damage response (DDR). Combining chemotherapy with inhibition of specific DDR pathways could therefore improve treatment efficacy. Multiple DDR pathways have been implicated in removal of platinum-DNA lesions, but it is unclear which exact pathways are most important to cellular platinum drug resistance. Here, we used CRISPR/Cas9 screening to identify DDR proteins that protect colorectal cancer cells against the clinically applied platinum drug oxaliplatin. We find that besides the expected homologous recombination, Fanconi anemia and translesion synthesis pathways, in particular also transcription-coupled nucleotide excision repair (TC-NER) and base excision repair (BER) protect against platinum-induced cytotoxicity. Both repair pathways are required to overcome oxaliplatin- and cisplatin-induced transcription arrest. In addition to the generation of DNA crosslinks, exposure to platinum drugs leads to reactive oxygen species production that induces oxidative DNA lesions, explaining the requirement for BER. Our findings highlight the importance of transcriptional integrity in cells exposed to platinum drugs and suggest that both TC-NER and BER should be considered as targets for novel combinatorial treatment strategies

Repair protein persistence at DNA lesions characterizes XPF defect with Cockayne syndrome features

The structure-specific ERCC1-XPF endonuclease plays a key role in DNA damage excision by nucleotide excision repair (NER) and interstrand crosslink repair. Mutations in this complex can either cause xeroderma pigmentosum (XP) or XP combined with Cockayne syndrome (XPCS-complex) or Fanconi anemia. However, most patients carry compound heterozygous mutations, which confounds the dissection of the phenotypic consequences for each of the identified XPF alleles. Here, we analyzed the functional impact of individual pathogenic XPF alleles on NER. We show that XP-causing mutations diminish XPF recruitment to DNA damage and only mildly affect global genome NER. In contrast, an XPCS-complex-specific mutation causes persistent recruitment of XPF and the upstream core NER machinery to DNA damage and severely impairs both global genome and transcription-coupled NER. Remarkably, persistence of NER factors at DNA damage appears to be a common feature of XPCS-complex cells, suggesting that this could be a determining factor contributing to the development of additional developmental and/or neurodegenerative features in XP patients

DNA damage sensitivity of SWI/SNF-deficient cells depends on TFIIH subunit p62/GTF2H1

Mutations in SWI/SNF genes are amongst the most common across all human cancers, but efficient therapeutic approaches that exploit vulnerabilities caused by SWI/SNF mutations are currently lacking. Here, we show that the SWI/SNF ATPases BRM/SMARCA2 and BRG1/SMARCA4 promote the expression of p62/GTF2H1, a core subunit of the transcription factor IIH (TFIIH) complex. Inactivation of either ATPase subunit downregulates GTF2H1 and therefore compromises TFIIH stability and function in transcription and nucleotide excision repair (NER). We also demonstrate that cells with permanent BRM or BRG1 depletion have the ability to restore GTF2H1 expression. As a consequence, the sensitivity of SWI/SNF-deficient cells to DNA damag

Polymorphisms in mirna-binding sites of nucleotide excision repair genes and colorectal cancer risk

Reduced DNA repair capacity and DNA damage accumulation may lead to cancer development. Regulation of and coordination between genes involved in DNA repair pathways is fundamental for maintaining genome stability, and post-transcriptional gene regulation by microRNAs (miRNAs) may therefore be of particular relevance. In this context, the presence of single nucleotide polymorphisms (SNPs) within the 3'untranslated regions of target DNA repair genes could alter the binding with specific miRNAs, modulating gene expression and ultimately affecting cancer susceptibility.In this study, we investigated the role of genetic variations in miRNA-binding sites of nucleotide excision repair (NER) genes in association with colorectal cancer (CRC) risk. From 28 NER genes, we screened among SNPs residing in their 3'untranslated regions and simultaneously located in miRNA-binding sites, with an in silico approach. Through the calculation of different binding free energy according to both alleles of identified SNPs, and with global binding free energies median providing a threshold, we selected nine NER gene variants. We tested those SNPs in 1098 colorectal cancer cases and 1469 healthy controls from the Czech Republic.Rs7356 in RPA2 and rs4596 in GTF2H1 were associated with colorectal cancer risk. After stratification for tumor location, the association of both SNPs was significant only for rectal cancer (rs7356: OR 1.52, 95% CI 1.02-2.26, P = 0.04 and rs4596: OR 0.69, 95% CI 0.50-0.94, P = 0.02; results not adjusted for multiple testing).Variation in miRNA target binding sites in the 3' untranslated region of NER genes may be important for modulating colorectal cancer risk, with a different relevance according to tumor location. © The Author 2012. Published by Oxford University Press. All rights reserved