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Multiple interaction nodes define the postreplication repair response to UV-induced DNA damage that is defective in melanomas and correlated with UV signature mutation load Author Pavey, S, Pinder, A, Fernando, W, D’Arcy, N, Matigian, N, Skalamera, D, Lê Cao, KA, Loo-Oey, D, Hill, MM, Stark, M, Kimlin, M, Burgess, A, Cloonan, N, Sturm, RA, Gabrielli, B Published 2019 Journal Title Molecular Oncology DOI https://doi.org/10.1002/1878-0261.12601 Copyright Statement © 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Downloaded from http://hdl.handle.net/10072/390173 Griffith Research Online https://research-repository.griffith.edu.au Multiple interaction nodes define the postreplication repair response to UV-induced DNA damage that is defective in melanomas and correlated with UV signature mutation load Sandra Pavey1, Alex Pinder1, Winnie Fernando2, Nicholas D’Arcy2, Nicholas Matigian1,3, Dubravka Skalamera1,2, Kim-Anh Le^ Cao1*, Dorothy Loo-Oey1, Michelle M. Hill1,4 , Mitchell Stark1 , Michael Kimlin5, Andrew Burgess6, Nicole Cloonan4†, Richard A. Sturm1 and Brian Gabrielli1,2 1 Diamantina Institute, TRI, The University of Queensland, Woolloongabba, QLD, Australia 2 Mater Research, TRI, The University of Queensland, Woolloongabba, QLD, Australia 3 QFAB Bioinformatics, The University of Queensland, Brisbane, QLD, Australia 4 QIMR Berghofer Medical Research Institute, Herston, QLD, Australia 5 University of the Sunshine Coast, Sippy Downs, QLD, Australia 6 ANZAC Research Institute, Concord, NSW, Australia Keywords Ultraviolet radiation-induced DNA mutations are a primary environmental DNA repair; G2 phase checkpoint; MASTL; driver of melanoma. The reason for this very high level of unrepaired postreplication repair; ultraviolet radiation DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV-induced lesions, that is, the nucleo- Correspondence B. Gabrielli, Mater Research, Translational tide excision repair pathway, appears intact in most melanomas. We have Research Institute, The University of previously reported a postreplication repair mechanism that is commonly Queensland, Brisbane, QLD 4102, Australia defective in melanoma cell lines. Here we have used a genome-wide Tel: +61734437641 approach to identify the components of this postreplication repair mecha- E-mail: [email protected] nism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally Present address assessed these to identify novel components of this repair and cell cycle *School of Mathematics and Statistics and Melbourne Integrative Genomics, The checkpoint network. We have identified multiple interaction nodes, includ- University of Melbourne, Melbourne, Vic., ing global genomic nucleotide excision repair and homologous recombina- Australia tion repair, and previously unexpected MASTL pathway, as components †School of Biological Science, The of the response. Finally, we have used bioinformatics to assess the contri- University of Auckland, New Zealand bution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of (Received 5 September 2019, revised 6 the pathway, especially the DNA damage repair components, are signifi- November 2019, accepted 14 November 2019, available online 19 December 2019) cant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signa- doi:10.1002/1878-0261.12601 ture mutation load. Abbreviations ATM, ataxia telangiectasia mutated; ATR, ATM and RAD3-related; CHK1, checkpoint kinase 1; GG-NER, global genomic NER; HR, homologous recombination; NER, nucleotide excision repair; PDS, Pathway Deregulation Score; RNA-seq, RNA sequencing; RPA, replication protein A; ssDNA, single-stranded DNA; TCGA, The Cancer Genome Atlas; TLS, translesion synthesis; UV, ultraviolet radiation; UV-G2 checkpoint, G2 phase checkpoint and postreplication repair response; UVR, UV radiation. 22 Molecular Oncology 14 (2020) 22–41 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. S. Pavey et al. Defective DNA repair mechanism in melanoma gap filling (Chang and Cimprich, 2009; Waters et al., 1. Introduction 2009; Wigan et al., 2012). How these diverse compo- Ultraviolet radiation (UVR) is the major environmen- nents function in this checkpoint and repair response tal mutagen driving the development of melanoma. are unclear. The genotoxic effect of UVR characteristically results Here, we have taken a genome-wide approach to in single-stranded DNA lesions, specifically 6–4 photo- investigate this coupled G2 phase checkpoint and products and cyclobutane pyrimidine dimers (Pavey postreplication repair response (UV-G2 checkpoint). et al., 2013). Melanomas commonly have very high We have analyzed the mRNAs differentially loaded numbers of mutations that are the direct consequence onto polyribosomes (polysome profiling) after UVR of unrepaired UV-induced lesions (Hodis et al., 2012). exposure to identify differentially translated proteins. UVR-damaged DNA bases are typically repaired by This approach was used to bridge the gap between nucleotide excision repair (NER) (Mouret et al., 2008; gene expression (i.e., total mRNA RNA-seq and Pavey et al., 2013). However, if the UV damage is not microarray analysis) and proteomics analysis, as only repaired before the cells undergo DNA replication, it a proportion of variation in protein abundance is can result in what has been collectively termed UV sig- explained by variations in mRNA levels (de Sousa nature mutations (Helleday et al., 2014). A specific Abreu et al., 2009). We have functionally validated a mutational signature (Signature 7) secondary to unre- large number of differentially polysome-loaded paired UVR damage has been found to be highly mRNAs using a high-throughput approach to under- prevalent in melanomas and involves a high propor- stand the complexity of this response to UVR and tion of dinucleotide C>T mutations (Alexandrov et al., identified several nodes that interact in this response. 2013). Finally, we have used pathway expression analysis to Although NER has a major role in repairing UVR show that dysregulation of this pathway is associated damage, it is unclear as to how common NER defects with increased UV signature mutations in melanoma. are in melanomas (Belanger et al., 2014; Budden et al., 2016; Gaddameedhi et al., 2010). A recent bioinfor- 2. Methods matic analysis of the TGCA melanoma dataset failed to find any correlation between dysregulated expression of NER pathway genes and the UV signature 2.1. Cell culture mutation load (D’Arcy et al., 2019). We have previously reported a G2 phase cell cycle A2058 and MM576 human melanoma cell lines were checkpoint-coupled repair mechanism that is triggered cultured as described previously (Wigan et al., 2012). in response to suberythemal doses of UVR in melano- All cell lines were confirmed to be mycoplasma free. cytes and keratinocytes in the epidermis (Pavey et al., Synchronized cell populations and UV irradiation and 2001). The G2 phase checkpoint is commonly defective UV-G2 checkpoint arrested populations were obtained in melanomas and results in cells accumulating as previously described (Wigan et al., 2012). For cellu- increased UV signature mutations after irradiation lar DNA content analysis, floating and adhered cells (Pavey et al., 2013; Wigan et al., 2012). This mecha- were collected and fixed in 70% ethanol at À20 °C nism repairs the small number of lesions remaining and analyzed by flow cytometry using a FACSCalibur after NER has repaired the bulk of lesions in G1 system (BD Biosciences, San Jose, CA, USA). Data phase. The checkpoint-coupled repair mechanism uti- analysis was performed using FLOWJO X software (Bec- lizes the replication fork to interrogate the entire gen- ton Dickson, Ashland, OR, USA). ome during replication to detect UV-induced lesions and repair them in G2 phase (Wigan et al., 2012). The 2.2. Immunoblotting G2 phase checkpoint is triggered by the presence of single-stranded DNA (ssDNA) that recruits RPA and Cells were lysed and analyzed by immunoblotting as the cell cycle checkpoint kinase ATR that activates described previously (Wigan et al., 2012). Antibodies CHK1 to impose a G2 phase cell cycle arrest until the against DDB1, POLG (LS Bioscience, Seattle, WA, ssDNA gaps are repaired. A suite of DNA repair pro- USA), NCOR2/SMRTE, SCRIB (Santa Cruz Biotech- teins colocalize with the RPA foci including BRCA1, nology, Dallas, TX, USA), XPC (GeneTex, Irvine, RAD51, and RAD18, suggesting the involvement of CA, USA), SMARCA4, MTAP (Abcam, Cambridge, homologous recombination (HR) and/or translesion UK), STAT3 (Becton Dickson, North Ryde, NSW, synthesis (TLS) Y family polymerase mechanism in the Australia), GDF15 (R&D Systems, Minneapolis, MN, Molecular Oncology 14 (2020) 22–41 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 23 Defective DNA repair mechanism in melanoma S. Pavey et al. USA), MASTL

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