Repair of Oxidatively Damaged Guanines

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Reactive oxygen species (ROS) are commonly generated as by-products during metabolism, during response to inflammation, and following exposure to ionizing radiation and chemicals. Oxidative DNA damage presents a serious challenge to genomic integrity and can accelerate carcinogenesis and aging. ROS-dependent DNA damage includes strand breaks and oxidative base lesions that are repaired by base excision repair (BER), initiated with removal of base lesions by DNA glycosylases. The overall goal of this project is to study the role of selected DNA glycosylases and mismatch repair enzymes in response to cellular oxidative stress. We focus on the role of human MutY homolog (hMYH) glycosylase and its interactions with other repair mechanisms. hMYH reduces stress-induced mutagenesis by removing misincorporated adenines paired with 8-oxoG (the most abundant form of DNA damage), therefore reduces G:C to T:A mutations. hMYH deficiency predispose individuals to colon cancer. hMYH interacts with the DNA replication machinery, other repair enzymes, the 9-1- 1 cell cycle checkpoint complex (Rad9/Rad1/Hus1), and SIRT6 which is implicated in regulation of aging. In this context, the 9-1-1 proteins interact with and increase the activity of hMYH glycosylase, hNEIL1 glycosylase, hOGG1 glycosylase, and hMSH2/hMSH6 mismatch recognition complex. We hypothesize that MYH and other DNA repair enzymes serve as molecular adaptors to recruit checkpoint proteins to DNA lesion sites and coordinate DNA repair and increase repair efficiency and fidelity. To examine this hypothesis, we propose the following specific aims: (1) The dynamic interactions of MYH with OGG1, NEIL1, and MSH2/MSH6 both in vitro and in vivo will be delineated. We will test whether these interactions are altered following oxidative stress and during the progression of the cell cycle. (2) The physical and functional Interactions of MYH (and Neil1, hOGG1 and hMSH2/hMSH6) with the 9-1-1 complex will be elucidated. We will investigate how different proteins compete for the 9-1-1 complex and why different glycosylases select different subunits of the 9-1-1 complex. The biological significance of Hus1-MYH interaction will be investigated by interruption of the interaction by mutagenesis and by using a Hus1 binding competitor peptide. We will test a model that DNA glycosylases act as adaptors to recruit the 9-1-1 complex to the lesion sites. (3) The role of an aging regulator SIRT6 in BER by stimulating MYH, but inhibiting NEIL1 activity will be studied. Because SIRT6 is required to regulate genomic integrity and impacts the aging process, revealing the mechanism of SIRT6 interaction in BER is important. Successful completion of these studies will reveal important new information regarding the interactions among DNA repair proteins, cell cycle checkpoints, and an aging regulating protein. We anticipate that these studies will advance our understanding of carcinogenesis process and form the background work for the development of new anti-cancer drugs.