Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context.
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.
Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010; Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Amanda
A
Gunn
Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010; Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Nicole
N
Bennardo
Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Anita
A
Cheng
Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010; Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010. Electronic address: jstark@coh.org.
Jeremy M
JM
Stark
D017766:Acid Anhydride Hydrolases; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002875:Chromosomes / Q000235:genetics*; D004247:DNA / Q000737:chemistry*; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000737:chemistry*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes