Abstract
Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3.
Highlights
In higher eukaryotes, DNA double strand breaks (DSBs) are predominantly repaired by a simple end joining process mediated by ligation that operates without homology requirements and is termed non-homologous end joining (NHEJ) [1,2,3]
dose equivalent (Deq) is the equivalent dose for remaining DSBs calculated from the fraction of DNA released (FDR) after each repair time interval on the basis of a dose-response curve obtained from the same population of cells processed immediately after irradiation
Together with our results on the ligation requirements of DT40 DNA replication [18] and results recently published in other cell systems [14,15,16], they confirm the restricted function of LIG4 in D-NHEJ and demonstrate a remarkable functional flexibility for LIG1 and LIG3 in semi-conservative DNA replication, and in B-NHEJ
Summary
DNA double strand breaks (DSBs) are predominantly repaired by a simple end joining process mediated by ligation that operates without homology requirements and is termed non-homologous end joining (NHEJ) [1,2,3]. NHEJ is associated with additions or deletions of nucleotides at the junction that alter the genome leaving ‘‘scars’’ behind [1]. Sequence preservation, when it occurs, is fortuitous and observed only for certain types of ‘‘clean’’ DNA ends generated by restriction endonucleases - it is unlikely for the chemically complex, modified ends generated by ionizing radiation (IR). We opt for the term DNA-PKcsdependent (D-NHEJ) for this pathway to emphasize the significance of this kinase in its evolutionary development [2,6]
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