Great progress has been made in uncovering the proteins and pathways that function in the replication stress response. In particular, the hereditary breast cancer genes, as well as genes mutated in Fanconi anemia (FA) function in the replication stress response. It is now understood that loss of their function in the replication stress response contributes to the sensitivity of associated tumors to chemotherapies, such as cisplatin. However, the distinct functions for the BRCA-FA proteins are largely unknown. Here, we propose to analyze DNA replication fork dynamics, replisome components, and identify patient mutations that have specific defects in the replication stress response. To define how a cell transitions from defective to dysregulated replication, we have engineered cells expressing different mutant versions of the BRCA1-associated FANCJ also mutated in breast/ovarian cancer and FA. Similar to BRCA1, we have uncovered that FANCJ functions to protect replication forks from collapse. We also found that this FANCJ fork protection function requires its direct interaction with the mismatch repair (MMR) protein, MLH1. This finding provides insight as to why cells lacking the FANCJ-MLH1 interaction fail to recover from replication stress. We have also identified putative gain-of-function FANCJ mutants, such as the BRCA1-interaction defective mutant, that circumvent replication stress, keep forks intact, and confer hyper- resistance to replication stress inducing agents.
In Aim 1, we will seek to define how FANCJ interactions direct DNA replication fork dynamics. Given that FANCJ localizes to replication forks, displaces proteins, and unwinds DNA, we hypothesize that disrupted vs dysregulated replication will reflect not only changes in DNA structures, but also the proteins found at DNA replication forks.
In Aim 2, we will seek to determine how FANCJ contributes to the composition of the replisome in both unchallenged and at stressed replication forks. Replication stress induces changes to FANCJ protein interactions and post-translation modifications. Some of these changes occur at sites we found to be mutated in cancer patients.
In Aim 3, we will seek to generate FANCJ mutants resistant to replication stress induced changes to uncover mechanisms regulating FANCJ function that are lost in cancer. Collectively, by defining how cells succumb to- or survive- toxic DNA damage that normally interferes with replication, we will gain insight towards mechanisms transitioning cells from defective to dysregulated replication in cancer. !

Public Health Relevance

The hereditary breast and ovarian cancer genes function along with many other DNA repair proteins to ensure that cells recover from replication stress without stalled DNA replication forks unraveling into DNA structures that drive genomic instability. Although tremendous progress has been made in understanding the replication stress response, its regulation by the BRCA-FA pathway, how perturbed in cancer, and targeted by chemotherapies, it remains to be fully characterized. Here, we propose to implement state-of-the-art assays to analyze DNA replication fork dynamics, replisome components, and identify patient mutations that have specific defects in the replication stress response in order to define mechanisms as to how the replication stress responses is coordinated and dysregulated in cancer. !

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Witkin, Keren L
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University of Massachusetts Medical School Worcester
Anatomy/Cell Biology
Schools of Medicine
United States
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