Abstract
During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3' nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing.
Highlights
Incorrect end use during repair can cause chromosome rearrangements
A double strand breaks (DSBs) Downstream from an Active Promoter Is More Reliant on ataxia telangiectasia-mutated kinase (ATM) for Limiting Distal End Use—Because the frequency of distal end use is higher when a DSB is downstream from an active promoter, we considered the possibility that the relative requirement for individual factors during end use could be affected by DSB transcription context
By examining a reporter system for end joining (EJ) repair of two tandem chromosomal DSBs, we present evidence that a number of DNA damage response factors are important for maintaining correct end use (i.e. ATM, DNA-PKcs, and RAD50), that the transcription context of a DSB can affect the frequency of correct end use, and that a DSB downstream from an active promoter shows a greater reliance on ATM for correct end use
Summary
Incorrect end use during repair can cause chromosome rearrangements. Results: DNA-PKcs and RAD50 limit incorrect end use, and a break downstream from an active promoter shows elevated incorrect end use; these factors and conditions have distinct effects on repair requiring end processing. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing. We have examined the role of individual DNA damage response factors and chromosomal break transcription context on the frequency of incorrect end use during EJ of two tandem DSBs, as well as the frequency of repair events requiring end processing. We present evidence that inhibition of DNA-PKcs kinase activity, or depletion of RAD50, each cause an elevated frequency of incorrect (distal) end use during repair of tandem DSBs. In contrast, we find that these factors have opposing effects on a homologous repair event that involves extensive DSB end processing (single strand annealing, SSA), in that DNA-PKcs inhibits SSA, whereas RAD50 promotes this repair event. We suggest that the roles of factors or conditions in maintaining correct end use during EJ cannot be predicted by their influence on repair that requires DSB end processing, such that these may be distinct aspects of repair
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