title: Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.
abstract: Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.|Department of Biochemistry and Molecular Biology, University of Texas Medical Branch (UTMB), Galveston, TX 77555, USA.
authors.forename: Arijit
authors.initials: A
authors.lastname: Dutta
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.|Texas A&M Health Science Center, College of Medicine, Bryan, TX 77807, USA.
authors.forename: Bradley
authors.initials: B
authors.lastname: Eckelmann
authors.affiliation: EntroGen, Inc., Woodland Hills, CA 91367, USA.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Adhikari
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.
authors.forename: Kazi Mokim
authors.initials: KM
authors.lastname: Ahmed
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.|Weill Cornell Medical College, New York, NY 10065, USA.
authors.forename: Shiladitya
authors.initials: S
authors.lastname: Sengupta
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.
authors.forename: Arvind
authors.initials: A
authors.lastname: Pandey
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.
authors.forename: Pavana M
authors.initials: PM
authors.lastname: Hegde
authors.affiliation: Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA 94720, USA.
authors.forename: Miaw-Sheue
authors.initials: MS
authors.lastname: Tsai
authors.affiliation: Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA 94720, USA.|Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.|Weill Cornell Medical College, New York, NY 10065, USA.|Houston Methodist Neurological Institute, Houston, TX 77030, USA.
authors.forename: Muralidhar L
authors.initials: ML
authors.lastname: Hegde
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.|Department of Biochemistry and Molecular Biology, University of Texas Medical Branch (UTMB), Galveston, TX 77555, USA.|Texas A&M Health Science Center, College of Medicine, Bryan, TX 77807, USA.|Weill Cornell Medical College, New York, NY 10065, USA.
authors.forename: Sankar
authors.initials: S
authors.lastname: Mitra
mesh_terms: D047390:Casein Kinase II / Q000378:metabolism*; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D014965:X-Rays; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D047390:Casein Kinase II