title: XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.
abstract: XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, East Sussex, BN1 9RQ, UK.
authors.forename: Enriqueta
authors.initials: E
authors.lastname: Riballo
authors.forename: Lisa
authors.initials: L
authors.lastname: Woodbine
authors.forename: Thomas
authors.initials: T
authors.lastname: Stiff
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Walker
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Goodarzi
authors.forename: Penny A
authors.initials: PA
authors.lastname: Jeggo
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005047:Etoposide / Q000633:toxicity; D006801:Humans; D051379:Mice; D009353:Zinostatin / Q000633:toxicity
chemical_list: D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D000249:Adenosine Monophosphate; D005047:Etoposide; D000255:Adenosine Triphosphate; D009353:Zinostatin; D011088:DNA Ligases; D000072481:DNA Ligase ATP