ORIGINAL ARTICLE K63-Ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair JL Metcalf 1 , PS Bradshaw 2 , M Komosa 3 , SN Greer 1 , M Stephen Meyn 2,3,4 and M Ohh 1 DNA repair is essential for maintaining genomic stability, and defects in this process significantly increase the risk of cancer. Clear- cell renal cell carcinoma (CCRCC) caused by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is characterized by high genomic instability. However, the molecular mechanism underlying the association between the loss of VHL and genomic instability remains unclear. Here, we show that suppressor of cytokine signaling 1 (SOCS1) promotes nuclear redistribution and K63-ubiquitylation of VHL in response to DNA double-strand breaks (DSBs). Loss of VHL or VHL mutations that compromise its K63-ubiquitylation attenuates the DNA-damage response (DDR), resulting in decreased homologous recombination repair and persistence of DSBs. These results identify VHL as a component of the DDR network, inactivation of which contributes to the genomic instability associated with CCRCC. Oncogene (2014) 33, 1055–1065; doi:10.1038/onc.2013.22; published online 4 March 2013 Keywords: DNA repair; K63-ubiquitylation; RCC; SOCS1; VHL INTRODUCTION DNA double-strand breaks (DSBs) are highly toxic lesions that if left unrepaired promote genomic instability, one of the hallmarks of cancer. To minimize the deleterious effects of DSBs, multi- cellular organisms utilize a complex signaling network known as the DNA-damage response (DDR) to detect the presence of DNA damage and activate appropriate cellular defences. In response to DSBs, ataxia–telangiectasia mutated (ATM) is rapidly autopho- sphorylated and recruited to DNA lesions by the Mre11–Rad50– Nbs1 (MRN) sensor complex. 1,2 Activated ATM phosphorylates histone variant H2AX (known as gH2AX when phosphorylated) surrounding the DSB, which promotes the recruitment of DDR proteins to the DNA lesion. 3,4 ATM also phosphorylates the effector kinases Chk1/2, which initiate a downstream signaling cascade through the phosphorylation of other DDR proteins, including cdc25, breast and ovarian cancer susceptibility protein (BRCA) 1, replication protein A (RPA), and p53. 5 Through these and other molecular events, the DDR promotes survival and genome stability by activating cell cycle checkpoints, initiating DNA repair and triggering stress responses. Alternatively, if the damage is too extensive or the DNA break irreparable, the DDR can induce apoptosis or senescence. 6,7 Non-homologous end joining and homologous recombination (HR) are the two major DSB repair pathways in eukaryotic cells. Non-homologous end joining directly ligates the DNA ends and is considered error-prone, as it frequently results in small deletions and translocations due to non-compatible end joining. In contrast, HR typically uses homologous DNA on a sister chromatid as a template for accurate repair and is generally restricted to S and G2 phases of the cell cycle. These mechanistically distinct pathways have both competitive and compensatory roles in DNA repair in an attempt to maintain genomic stability. 8–13 Mutations in DDR genes have pleiotropic effects on cell behavior and genome stability and have been linked to many sporadic and inherited forms of cancer. 14 For example, homozygous mutations in ATM cause a childhood disease called ataxia–telangiectasia, which is characterized by genomic instabil- ity, immunodeficiency, neurodegeneration, radiation hypersensi- tivity and a predisposition to cancer. 15 Similarly, mutations in the core MRN sensor complex proteins, Mre11 and Nbs1, lead to ataxia–telangiectasia-like disorder and Nijmegen breakage syn- drome, respectively, which share the radio-sensitivity and genomic instability associated with ataxia–telangiectasia. 16 BRCA1 and BRCA2 have been shown to have essential roles in HR and individuals with mutations in these genes are at increased risk of developing breast and ovarian cancer. 17 Clear-cell renal cell carcinoma (CCRCC), the most common form of kidney cancer and arguably one of the most aggressive forms of cancer, is associated with high genomic instability. Aberrations of chromosome 3 with loss of 3p or translocations of different chromosome sections to the deleted chromosome 3 regions are most common. 18 Gain of chromosomes 5q and 7 are also frequent and additional loss of 1p, 4, 9, 13q and 14q has been associated with a more aggressive disease. 18 Although the molecular mechanisms underlying this genomic instability have remained unclear, mutations in the SWI/SNF chromatin remodeling complex gene, PBRM1, were recently identified in B41% of CCRCC cases. 19 In addition, mutations in the histone- modifying genes UTX, JARID1C and SETD2 have been identified in a small number of cases. 19 These recent findings suggest that changes in chromatin structure have an important role in CCRCC tumourigenesis and may underlie some of the genomic instability. However, the majority of CCRCC cases have no reported mutations in chromatin remodeling or DNA repair genes, and the molecular 1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 2 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; 3 Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada and 4 Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada. Correspondence: Professor M Ohh, Department of Laboratory Medicine and Pathobiology, University of Toronto, Medical Sciences Building, Room 6306, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: michael.ohh@utoronto.ca Received 2 July 2012; revised 17 December 2012; accepted 11 January 2013; published online 4 March 2013 Oncogene (2014) 33, 1055–1065 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc