The long-term goal of this AREA project focuses on the molecular mechanisms of DNA damage checkpoint and DNA replication, which has significant implications for the study of biological and pathological processes such as cancer, aging, immune deficiency and neurodegenerative disorders. Genomes of all living organisms are exposed to a variety of threats, but the genome's DNA damage checkpoint functions as a surveillance mechanism, monitoring damage in a genome and then directly cellular responses through sensor, mediator, and effector proteins to resolve the problem. If a checkpoint fails to activate when necessary, the result is unrepaired damage that leads to genomic instability and tumor formation. The main barriers to understanding this process are in identifying how checkpoints sense DNA damage and how the proteins relay the damage signal. Specifically, it is not known how the activated sensor kinase ATR phosphorylates its downstream effector protein Chk1. In addition to the critical roles in DNA replication initiation and DNA replication stress response, TopBP1 plays an important role in DNA damage checkpoint signaling through its C-terminus region. However, the underlying mechanism is not known. Preliminary data in Xenopus egg extracts show that WDR18, a TopBP1-interacting protein, interacts with TopBP1 C-terminus, and their interaction is required for ATR activation of Chk1 in the response to double-stranded breaks. The goal of this R15 project is to test the hypothesis that TopBP1 partner WDR18 plays essential roles in DNA damage checkpoint and DNA replication. This R15 project will be carried out in Xenopus egg extracts, a reliable cell-free biochemical system, that has been used for a wide variety of studies in cell cycle, DNA replication, and checkpoint activation.
The specific aims are: (1) To elucidate the role of WDR18, a TopBP1 partner, in DNA damage checkpoint signaling in Xenopus egg extract through various biochemical and molecular biology approaches;and (2) To determine if WDR18 is required for DNA replication under normal or stressful conditions, by monitoring bulk DNA synthesis product with the presence or absence of damaging agents when endogenous WDR18 is removed by immunodepletion. These studies will identify a novel checkpoint and replication protein, and will enable a better understanding of how a DNA damage checkpoint works to protect genomic integrity and how cancer develops when DNA damage is not repaired or not triggering appropriate responses.
Project Narrative Accurate duplication of genetic information and sensing any possible DNA damage by a surveillance mechanism DNA damage checkpoint are vital for the maintenance of genomic integrity and tumor suppression. We will use a simple model system, Xenopus egg extracts derived from frog eggs, to examine how DNA damage checkpoint signaling is initiated and relayed, and how DNA is copied with high fidelity. Revealing the details of these essential processes will provide a better understanding of how cancer develops and ultimately novel avenues to design anti-cancer drugs by manipulating novel checkpoint and replication proteins.
|Yan, Shan; Sorrell, Melanie; Berman, Zachary (2014) Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress. Cell Mol Life Sci 71:3951-67|
|Bai, Liping; Michael, W Matthew; Yan, Shan (2014) Importin ?-dependent nuclear import of TopBP1 in ATR-Chk1 checkpoint in Xenopus egg extracts. Cell Signal 26:857-67|
|Yan, Shan; Willis, Jeremy (2013) WD40-repeat protein WDR18 collaborates with TopBP1 to facilitate DNA damage checkpoint signaling. Biochem Biophys Res Commun 431:466-71|
|Willis, Jeremy; Patel, Yogin; Lentz, Barry L et al. (2013) APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress. Proc Natl Acad Sci U S A 110:10592-7|