Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency
Significance Chromosomal rearrangements, such as large deletions, can cause loss of tumor suppressor genes. Rearrangements can be formed by aberrant end-joining (EJ) repair that ligates ends from different double-strand breaks. Two distinct EJ pathways could potentially cause rearrangements [canonical nonhomologous end joining (C-NHEJ) and alternative EJ], which are mediated by different factors and cause distinct repair junction patterns. Using a reporter for a 0.4-Mbp deletion rearrangement, we found that cells deficient in the Ataxia Telangiectasia Mutated (ATM) signaling pathway show an increase in the frequency of this rearrangement and a shift toward C-NHEJ–mediated rearrangements. Thus, the relative contribution of distinct EJ pathways to rearrangement formation is affected by ATM signaling, which provides insight into the tumor suppressor function of ATM. A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3′ exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.
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