XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.

[1]  F. Alt,et al.  Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences. , 2005, Advances in immunology.

[2]  Alfonso Baldi,et al.  Global Gene Expression Profiling Of Human Pleural Mesotheliomas: Identification of Matrix Metalloproteinase 14 (MMP-14) as Potential Tumour Target , 2009, PloS one.

[3]  P. Frit,et al.  Alternative end-joining pathway(s): bricolage at DNA breaks. , 2014, DNA repair.

[4]  A. Fischer,et al.  Cernunnos, a Novel Nonhomologous End-Joining Factor, Is Mutated in Human Immunodeficiency with Microcephaly , 2006, Cell.

[5]  M. Lieber,et al.  Non-homologous end joining often uses microhomology: implications for alternative end joining. , 2014, DNA repair.

[6]  Yunmei Ma,et al.  Hairpin Opening and Overhang Processing by an Artemis/DNA-Dependent Protein Kinase Complex in Nonhomologous End Joining and V(D)J Recombination , 2002, Cell.

[7]  M. Sternberg,et al.  Protein structure prediction on the Web: a case study using the Phyre server , 2009, Nature Protocols.

[8]  N. Mailand,et al.  Assembly and function of DNA double-strand break repair foci in mammalian cells. , 2010, DNA repair.

[9]  Tom L. Blundell,et al.  PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair , 2015, Science.

[10]  S. Jackson,et al.  XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote DNA Nonhomologous End-Joining , 2006, Cell.

[11]  Jukka Westermarck,et al.  ColonyArea: An ImageJ Plugin to Automatically Quantify Colony Formation in Clonogenic Assays , 2014, PloS one.

[12]  B. Reina-San-Martin,et al.  PARP-3 and APLF function together to accelerate nonhomologous end-joining. , 2011, Molecular cell.

[13]  Xingzhi Xu,et al.  Protein Phosphatase 6 Interacts with the DNA-Dependent Protein Kinase Catalytic Subunit and Dephosphorylates γ-H2AX , 2010, Molecular and Cellular Biology.

[14]  S. Elledge,et al.  The DNA damage response: making it safe to play with knives. , 2010, Molecular cell.

[15]  Jiri Bartek,et al.  Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks , 2006, The Journal of cell biology.

[16]  F. Sturtz,et al.  Gene expression of HIF-1alpha and XRCC4 measured in human samples by real-time RT-PCR using the sigmoidal curve-fitting method. , 2007, BioTechniques.

[17]  Ulf Hellman,et al.  Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair. , 2011, Genes & development.

[18]  K. Caldecott,et al.  One ring to bring them all--the role of Ku in mammalian non-homologous end joining. , 2014, DNA repair.

[19]  Dale A Ramsden,et al.  Nonhomologous end joining: a good solution for bad ends. , 2014, DNA repair.

[20]  Yongbao Wang,et al.  A role for XLF in DNA repair and recombination in human somatic cells. , 2014, DNA repair.

[21]  Louise Fairall,et al.  A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase-8 Chain Assembly in Mediating Apoptotic Cell Death , 2012, Molecular cell.

[22]  Renata Walewska,et al.  Protein Profiling of Plasma Membranes Defines Aberrant Signaling Pathways in Mantle Cell Lymphoma* , 2009, Molecular & Cellular Proteomics.

[23]  M. Lieber,et al.  Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells , 1997, Nature.

[24]  David J. Chen,et al.  DNA-PK: a dynamic enzyme in a versatile DSB repair pathway. , 2014, DNA repair.

[25]  T. Blundell,et al.  The spatial organization of non-homologous end joining: From bridging to end joining , 2014, DNA repair.

[26]  Stefan Tenzer,et al.  Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics , 2013, Nature Methods.

[27]  J. Tainer,et al.  XLF regulates filament architecture of the XRCC4·ligase IV complex. , 2010, Structure.

[28]  P. Jeggo,et al.  Dna Repair , 2022 .

[29]  R. Ghirlando,et al.  Crystal structure of the Xrcc4 DNA repair protein and implications for end joining , 2000, The EMBO journal.

[30]  M. Gorenstein,et al.  Simultaneous Qualitative and Quantitative Analysis of theEscherichia coli Proteome , 2006, Molecular & Cellular Proteomics.

[31]  G. Chu,et al.  Crystal structure of human XLF: a twist in nonhomologous DNA end-joining. , 2007, Molecular cell.