The role of the checkpoint clamp in DNA repair

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Exposure to reactive oxygen species (ROS) and radiation leads to DNA damage that compromise genomic integrity. 8-oxo-guanine is one of the most frequent and highly mutagenic oxidative lesions because it mispairs with adenine during DNA replication. Oxidized base lesions are primarily eliminated by the base excision repair (BER) pathway. BER is tightly coordinated with DNA damage response (DDR) in order to maintain genomic stability and cell survival. Although BER and DDR have been well studied separately, the coordination of both processes is not understood. The heterotrimeric 9-1-1 (Rad9-Rad1-Hus1) checkpoint clamp plays dual roles in activation of DDR and DNA repair processes. We have identified unique interactions among BER and DDR proteins, demonstrating a novel and critical contribution of 9-1-1 to BER. We hypothesize that 9-1-1 provides a platform to coordinate BER processes to avoid the accumulation of toxic intermediates. The goal of this project is to define the biochemical and functional relationships between 9-1-1 and two enzymes that mediate initial steps of BER. The MYH/MUTYH DNA glycosylase excises misincorporated adenines paired with 8-oxo- guanine to prevent gene mutation in the first step of BER. APE1 endonuclease subsequently nicks DNA at abasic sites in the second step of BER. Studies of this coordination are important because these proteins are key players in processes as telomere maintenance and human disease prevention. It has been shown that mutations in human MYH gene are associated with colorectal cancer while APE1 and 9-1-1 are essential for cell viability and development. The following three specific aims are proposed. (1) We will define a functional DNA repair complex consisting of MYH, APE1, and 9-1-1. We will examine whether the formation of this repair complex is critical for their biological functions in maintaining genomic stability by interrupting the protein- protein interactions. (2) We will test a model that each subunit of 9-1-1 plays a distinct role in BER and DDR. We propose that Rad9 stabilizes BER machinery on DNA and that Hus1 promotes the smooth transfer of the toxic intermediate from MYH to APE1. (3) We will employ novel inducible ROS systems to confine DNA damage to a single genomic location and telomeres. We will use these systems to compare the BER rates and the order of BER factor association at both locations. We will examine the mutual dependence of BER factor association with sites of DNA damage using knockdown and knockout approaches. These studies will reveal exactly how the BER complex is assembled at lesion sites on fine scale and why BER is important for maintaining telomere integrity. The insights gained from these studies will significantly advance our understanding of the roles of BER and DDR in carcinogenesis, cancer treatment, and aging. Because DNA repair and 9-1-1 mediated signaling are associated with cancer development and treatment, interrupting these coordinated processes could provide an innovative strategy for the development of anticancer drugs.