title: A role for human homologous recombination factors in suppressing microhomology-mediated end joining.
abstract: DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Ahrabi
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.
authors.forename: Sovan
authors.initials: S
authors.lastname: Sarkar
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.
authors.forename: Sophia X
authors.initials: SX
authors.lastname: Pfister
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Pirovano
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.
authors.forename: Geoff S
authors.initials: GS
authors.lastname: Higgins
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W12 0NN, UK.
authors.forename: Andrew C G
authors.initials: AC
authors.lastname: Porter
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK timothy.humphrey@oncology.ox.ac.uk.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Humphrey
mesh_terms: D019313:BRCA1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D024682:BRCA2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001681:Biological Assay; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D000076228:MRE11 Homologue Protein; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D059767:Recombinational DNA Repair; D051716:Replication Protein A / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D000097462:DNA Polymerase theta
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein; D002352:Carrier Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins; D011065:Poly(ADP-ribose) Polymerases; D034741:RNA, Small Interfering; D051716:Replication Protein A; D000097462:DNA Polymerase theta; C492913:BRCA1 protein, human; C551750:BRCA2 protein, human; C121766:MRE11 protein, human; C497167:RPA1 protein, human; C410741:RBBP8 protein, human