Ionizing radiation (IR) can cause to significant tumor cell death, however resistance to radiotherapy can limit its efficacy. IR induces DNA double-strand breaks (DSBs) that can lead to toxic chromosomal rearrangements;such that limiting these rearrangements may be critical for radioresistance. Such IR-induced chromosomal rearrangements likely form via end joining (EJ) that uses incorrect DSB ends during repair. Thus, our long-term goal is to define the factors that limit rearrangements during EJ, and thereby develop therapeutic targets for tumor radiosensitization. To advance this goal, we developed a unique assay to quantify the use of correct versus incorrect ends during EJ of multiple DSBs. Using a chromosomal reporter with two tandem DSBs, and our technical innovation of generating site-specific non-cohesive DSBs, we can measure EJ that uses proximal ends that flank a single DSB versus EJ that uses distal ends of two DSBs. Distal end use during EJ is incorrect, since it causes a deletion rearrangement. Using this system, we found that the DNA damage response factors RAD50 and DNA-PKcs are important to limit incorrect end use during EJ. Since these factors also promote radioresistance, we propose to test our central hypothesis that the role of RAD50 and DNA-PKcs in limiting incorrect end use during EJ is critical for cellular radioresistance. A corollary of this hypothesis is that the funcion of these factors in limiting incorrect end use during EJ is a target for radiosensitization.
Aim 1 : To determine the importance to radioresistance of RAD50 function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. For this, we will examine a series of RAD50 mutants for the ability to complement a set of DNA repair functions, and promote radioresistance, in RAD50-deficient human cells.
Aim 2 : To determine the importance to radioresistance of DNA-PKcs function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. Using a similar approach as Aim 1, we will examine a series of DNA-PKcs mutants for the ability to complement DNA repair functions, and promote radioresistance, in DNA-PKcs-deficient mammalian cells.
Aim 3. To determine how increasing the distance between two tandem DSBs affects incorrect end use during EJ, and the relative requirement of RAD50 and DNA-PKcs for limiting such EJ-mediated deletion rearrangements. These proposed studies are significant, because they will provide novel insight into how RAD50 and DNA-PKcs promote radioresistance, which is critical for their development as therapeutic targets for tumor radiosensitization. Our study is innovative because it will establish a new paradigm for understanding the role of RAD50 and DNA-PKcs in promoting radioresistance, and because we propose in Aim 3 to develop a new reporter system to examine how the distance between DSBs affects end use during EJ. Our proposed study is also innovative because it will provide insight into the importance of incorrect end use during EJ for radiation toxicity, which will lead to new therapeutic strategies for tumor radiosensitization.
The proposed research is relevant to public health, because determining how DNA repair proteins limit radiation toxicity will lead to new therapeutic strategies for tumor radiosensitization. Thus, the proposed study supports the NIH mission for improving patient outcomes from cancer treatment.
|Onyango, David O; Lee, Gabriella; Stark, Jeremy M (2017) PRPF8 is important for BRCA1-mediated homologous recombination. Oncotarget 8:93319-93337|
|Bhargava, Ragini; Carson, Caree R; Lee, Gabriella et al. (2017) Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency. Proc Natl Acad Sci U S A 114:728-733|
|Onyango, David O; Howard, Sean M; Neherin, Kashfia et al. (2016) Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination. Nucleic Acids Res 44:5702-16|
|Morales, Maria E; Derbes, Rebecca S; Ade, Catherine M et al. (2016) Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes. PLoS One 11:e0151367|
|Skrdlant, Lindsey; Stark, Jeremy M; Lin, Ren-Jang (2016) Myelodysplasia-associated mutations in serine/arginine-rich splicing factor SRSF2 lead to alternative splicing of CDC25C. BMC Mol Biol 17:18|
|Kuo, Ching-Ying; Li, Xu; Stark, Jeremy M et al. (2016) RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner. Cell Cycle 15:787-98|
|Bhargava, Ragini; Onyango, David O; Stark, Jeremy M (2016) Regulation of Single-Strand Annealing and its Role in Genome Maintenance. Trends Genet 32:566-75|
|Howard, Sean M; Yanez, Diana A; Stark, Jeremy M (2015) DNA damage response factors from diverse pathways, including DNA crosslink repair, mediate alternative end joining. PLoS Genet 11:e1004943|
|Rein, Katrin; Yanez, Diana A; Terré, Berta et al. (2015) EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele. Nucleic Acids Res 43:7371-87|
|Muñoz, Meilen C; Yanez, Diana A; Stark, Jeremy M (2014) An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells. Nucleic Acids Res 42:7720-33|
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