title: Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes.
abstract: DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Anirban
authors.initials: A
authors.lastname: Chakraborty
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Nisha
authors.initials: N
authors.lastname: Tapryal
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Venkova
authors.affiliation: Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, Texas 77030, USA.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Horikoshi
authors.affiliation: Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, Texas 77030, USA.
authors.forename: Raj K
authors.initials: RK
authors.lastname: Pandita
authors.affiliation: Division of Life Sciences, Department of Cancer and DNA Damage Responses, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
authors.forename: Altaf H
authors.initials: AH
authors.lastname: Sarker
authors.affiliation: Department of Neurology and Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Partha S
authors.initials: PS
authors.lastname: Sarkar
authors.affiliation: Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, Texas 77030, USA.
authors.forename: Tej K
authors.initials: TK
authors.lastname: Pandita
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Hazra
mesh_terms: D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D057809:HEK293 Cells; D006801:Humans; D007763:Lac Operon; D010957:Plasmids; D012313:RNA / Q000235:genetics*; D012319:RNA Polymerase II / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D016914:Ribonuclease H / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D012313:RNA; D012319:RNA Polymerase II; D016914:Ribonuclease H