Abstract 3802: A novel uracil-DNA glycosylase, UdgX, as a new biochemical tool to directly detect uracils in DNA

Abstract

Abstract The enzyme activation-induced deaminase (AID) is essential to the B lymphocyte development and antibody maturation. It converts cytosines in DNA to uracil. The unregulated expression of AID in many B cell cancers is correlated with an increased load of mutations. AID also promotes DNA strand breaks and is required for chromosomal translocations such as IgH/Myc in these tumors. We have previously showed that B cell cancer cell lines and patient tumors which express AID at high levels also have high levels of uracil in their genomic DNA. We accomplished this by excising the uracils using a uracil-DNA glycosylase (Ung) and labeled the resulting in an abasic site with an aldehyde reactive chemical (ARP). ARP contains a biotin tag and it was detected using a fluorescently labeled streptavidin. The results from this assay showed B cell tumors have as much as 100-fold higher levels of genomic uracils. Further development of this assay has allowed the detection of uracils in the blood plasma and buffy coat of B cell cancer patients. This “liquid biopsy” assay technique has the potential to work as a simple surveillance tool for B cell cancer patients who are in remission. In addition, we are exploring the possibility of a direct detection of uracil in DNA, which would provide greater sensitivity to this assay and simplify it. A unique uracil-DNA glycosylase was recently isolated from M. smegmatis, UdgX, that binds covalently to uracils in DNA. We are developing multiple ways to detect uracil-DNA-UdgX complexes and amplifying the resulting signal. We expect that this will allow us to directly visualize uracils in B-NHL tumors and detect uracils in circulating B-NHL tumor DNA from a drop of patient plasma. Citation Format: Jessica Stewart, Shanqiao Wei, Madhurima Datta, Umesh Varshney, Ashok Bhagwat. A novel uracil-DNA glycosylase, UdgX, as a new biochemical tool to directly detect uracils in DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3802. doi:10.1158/1538-7445.AM2017-3802