title: Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.
abstract: Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response.
authors.affiliation: Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Edmonds
authors.affiliation: Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Carter
authors.affiliation: Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Nickson
authors.affiliation: Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK.
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Williams
authors.affiliation: Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK j.parsons@liverpool.ac.uk.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Parsons
mesh_terms: D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D011836:Radiation Tolerance / Q000235:genetics; D011839:Radiation, Ionizing; D011993:Recombinant Fusion Proteins; D000070584:Tripartite Motif Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases / Q000302:isolation & purification / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000070584:Tripartite Motif Proteins; D025521:Tumor Suppressor Proteins; C502830:HUWE1 protein, human; C519439:TRIM26 protein, human; D044767:Ubiquitin-Protein Ligases; D045647:DNA Glycosylases; C473634:NEIL1 protein, human