A systems-level analysis of DNA damage and repair in senescence, aging and cancer

About This Grant

Project Summary DNA damage has been implicated as driver of cell senescence and human disease, yet almost nothing is known about the true spectrum of DNA lesions that occur in cells and tissues. Similarly, defects in DNA repair are causally linked to human diseases such as premature aging and neurodegeneration, yet the actual substrates for DNA repair enzymes in vivo are unknown and have been assumed from in vitro studies. Complicating our understanding of DNA damage and repair are the potential for myriad highly reactive adduct-competent metabolites from endogenous and exogenous exposures and the emerging recognition of DNA adducts as epigenetic regulators of transcription. Here we propose an interdisciplinary collaboration bringing multiple convergent technologies to bear on defining the interplay between DNA adducts and DNA repair and specifically how this affects DNA replication stress, senescence, and cellular aging. In two aims, we propose studies to (1) lay the groundwork for defining the true spectrum of DNA adducts in human cells, (2) identify adducts that accumulate in senescing cells and drive replication stress, (3) assess how environmental exposures alter the adduct landscape and drive senescence, (4) identify DNA repair pathways for key subsets of these adducts, and (5) understand how loss of repair pathways drives cell senescence and phenotypes of aging. The proposed studies make use of mass spectrometric “adductomics” to discover and quantify DNA lesions and an in vivo host cell reactivation assay (FM-HCR) to measure the capacity of all major DNA repair pathways. As model cell systems, we created a library of DNA repair gene knockouts (KO) in RPE-1 retinal epithelial cells and IMR90 and BJ-1 fibroblasts, each lacking one of seven BER enzymes involved in initiating DNA repair at replication forks. We will apply these tools in two Specific Aims. In Aim 1, we will define repair pathways, replication stress signatures, and activation of the DNA damage response for endogenous and toxicant-induced DNA adducts. These studies compare endogenous sources of DNA damage and exposure to low doses of well-studied toxicants (MMS, H2O2) to test the idea of DNA repair enzymes as epigenetic regulators of gene expression and to define the physiological substrates of DNA repair enzymes. In Aim 2, we assess DNA repair capacity and adduct load as drivers of the senescence-associated secretory phenotype (SASP). These studies (1) determine if accumulation of DNA adducts during senescence is due to decreased DNA repair capacity or increased metabolism, (2) identify senescence-dependent DNA adducts, (3) quantify changes in replication stress during the evolution of senescence, and (4) quantify the effects of toxicant exposures on the senescence time course. The proposed studies provide the first glimpse into the broad spectrum of DNA adducts in human cells and the true substrates for DNA repair systems. In future studies, the results from these human cell models will be readily translated to DNA repair-deficient mouse models and to human tissues to discover disease-driving DNA adducts.