Genotoxic stress response and mutagenesis in normal tissues of mice deficient in homology directed repair

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Project Summary/Abstract The long-term objective of this project is to characterize the pathways and mechanisms that preserve viability and genome integrity in adult mammals exposed to environmental genotoxic stresses. DNA damage can be repaired by the high fidelity pathway homology directed repair (HDR) or by error prone non-homologous end joining (NHEJ). The consequences of HDR loss in adult vertebrates is not well understood because germline knockouts are embryonic lethal, thus, most of our understanding has come from cell culture models or inferred from tumors caused by HDR deficiency. To address this critical need, we have generated genetically engineered mice that can undergo inducible deletion of up to 99% of the essential HDR gene Brca1 in every tissue, post development. We will use this model to address critical questions on 1) the role of HDR in cell/tissue/organismal viability after different types of DNA damage; 2) the dependence of different tissues on HDR to maintain genome integrity after different types of DNA damage; 3) mutation signatures in tissues of HDR deficient mice caused by different stresses, allowing inference of repair pathway utilized in absence of HDR; 4) how the transcription factor p53 cooperates with HDR in each of these phenotypes; 5) the cooperative role of HDR and NHEJ in DNA repair. We hypothesize that in mice with deletion of the essential HDR gene Brca1, tissues known to develop HDR deficient tumors will have a higher mutation rate and favor p53 mediated programs of arrest over apoptosis compared to non-tumor prone tissues. Specific Aim 1. Determine how HDR preserves viability by mediating tissue specific cellular and transcriptional responses. In wild type or Brca1 deleted mice, we will examine how Brca1/HDR preserves near-term viability after different types of DNA damage by 3 measures: 1) the DNA damage response and cell fate (apoptosis, cell cycle arrest); 2) transcriptomic changes; 3) pathology of different tissues. Specific Aim 2. Determine tissue and DNA damage-specific role of Brca1/HDR in maintaining genome integrity. In this aim, we will examine long-term consequences of HDR deficiency on genome maintenance. Somatic mutations are known to accumulate in normal human tissues with age and are linked to disease. We will examine fidelity of repair and longevity of mice with or without functional HDR that survive different types of DNA damage. Deep sequencing and mutation signature analysis will reveal what tissues rely on HDR and what type of DNA perturbation is most mutagenic in different HDR deficient tissues. Specific Aim 3. Determine viability and stress response in mice deficient in NHEJ or both NHEJ and HDR. Because embryonic deletion of either Brca1 or the NHEJ gene Lig4 (coding for Ligase 4) is lethal, their individual and cooperative roles in adult tissues are not known. In this aim, we will examine survival and stress response after deletion of both HDR and NHEJ pathways in adult mice, leaving only SSA and alt-NHEJ.