Ionizing radiation (IR) is a clastogenic agent, in that IR exposure causes DNA double-strand breaks (DSBs). Failure to restore the original DNA sequence following clastogen exposure causes genetic loss, which likely contributes to the cellular toxicity of these agents. Clastogen-induced loss of genetic information can be a consequence of DSB repair that is non-restorative, as occurs with Single Strand Annealing (SSA) repair. Namely, SSA is a type of homologous recombination (HR) pathway that uses flanking homologous repeat sequences to bridge the DSB, which causes a deletion rearrangement, with loss of the genetic information between the repeats. SSA has the potential to be catastrophic to the integrity of mammalian genomes, given the high level of repetitive elements, including the approximately one million Alu-type elements in the human genome. In contrast, another HR pathway, Homology-Directed Repair (HDR), is relatively restorative, since it uses the precise sister chromatid as a template. Our overall hypothesis is that SSA protects against chromosome loss, but regulation of this pathway to favor HDR vs. SSA, and limit the size of deletion rearrangements caused by SSA, are important for clastogen resistance. We have identified two factors as being important for the regulation of SSA: the tetratricopeptide repeat protein XAB2 is a mediator of SSA, whereas the E3 ubiquitin ligase RNF168 inhibits SSA in a manner that is magnified in cells depleted of the HR mediator BRCA1. To understand the regulation of SSA, our specific aims are:
Aim 1. To define the SSA mediator function of XAB2. We will test the hypotheses that XAB2 promotes the end resection step of HR (HDR and SSA), and that motifs of XAB2 important for HR are critical for its interaction with another HR mediator, PRP19. We will also test the hypothesis that hypomorphic XAB2 mutants deficient for SSA can retain function to mediate the restorative HDR pathway, and hence also be proficient at promoting clastogen resistance.
Aim 2. To define the SSA inhibition function of RNF168. We will test the hypothesis that RNF168 functions in the same pathway as one SSA inhibitor (H2AX), but is distinct from another (Ku70, c-NHEJ pathway). Furthermore, we will test the hypothesis that the RNF168 C-terminus contains a negative regulatory domain that limits its anti-SSA activity.
Aim 3. To examine how the distance between DSBs and repeat sequences affects the frequency and regulation of SSA. For this, we will develop a novel reporter for repeat-mediated deletion rearrangements, which will also include variations in repeat sequence divergence. This is relevant to understanding genetic loss, since the DSB/repeat distance defines the size of the deletion caused by SSA. This project is significant, as it will uncover mechanisms that influence genetic loss caused by clastogen exposure. The innovation in this work lies in the novel insight into XAB2 and RNF168 function during genome maintenance, and a unique reporter system to examine repeat-mediated deletion rearrangements.

Public Health Relevance

The proposed research is relevant to public health, because determining how clastogen (e.g. radiation) exposure causes loss of genetic information will lead to new therapeutic strategies for tumor sensitization to therapeutic clastogens, as well as provide insight into the mechanisms that influence genetic loss caused by environmental clastogens. Thus, the proposed study supports the NIH mission for improving patient outcomes from cancer treatment, and understanding the causes of disease.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Radiation Therapeutics and Biology Study Section (RTB)
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Witkin, Keren L
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Beckman Research Institute/City of Hope
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Mendez-Dorantes, Carlos; Bhargava, Ragini; Stark, Jeremy M (2018) Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements. Genes Dev 32:524-536
Bhargava, Ragini; Sandhu, Manbir; Muk, Sanychen et al. (2018) C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains. Nat Commun 9:2484
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; Lee, Gabriella; Stark, Jeremy M (2017) PRPF8 is important for BRCA1-mediated homologous recombination. Oncotarget 8:93319-93337
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
Bhargava, Ragini; Onyango, David O; Stark, Jeremy M (2016) Regulation of Single-Strand Annealing and its Role in Genome Maintenance. Trends Genet 32:566-575