Abstract 2066: Measuring the activity of SMUG1 DNA glycosylase with a novel sSTRIDE-SMUG1 assay

Abstract On average, spontaneous deamination of cytosine to aberrant base uracil happens 70-200 times per human genome per day. These mutagenic U/G mispairs are, however, corrected by error-free base repair (BER) initiated by uracil-DNA glycosylases. SMUG1 (single-strand selective monofunctional uracil DNA glycosylase), as one of the major glycosylases, removes uracil and oxidized pyrimidines from DNA. In spite of the name, SMUG1 removes uracil also from double-stranded DNA. Unrepaired by SMUG1 U/G mispairs are mutagenic, and alterations in SMUG1 expression levels have been linked to cancer development. Low SMUG1 transcripts are associated with poor prognosis in breast cancer and SMUG1 loss has been shown to cause PARPi resistance in HR-deficient background. Hence, tools describing SMUG1 activities in DNA damage response may provide new approaches for cancer cell treatment. We report here the development, optimization and validation of an assay measuring the activity of SMUG1. The new assay is based on the STRIDE platform technology, which enables direct and sensitive detection of single- or double-strand DNA breaks in situ, in fixed cells. sSTRIDE-SMUG1 assay detects DNA nicks localized in close proximity to the SMUG1 protein and can thus be utilized as a direct reporter of its activity. The experiments were performed in HAP1 cells. First, in untreated cells we have shown that the sSTRIDE-SMUG1 signals constitute ca. 1-2% of total single-strand DNA breaks foci detected by the classic sSTRIDE assay variant, while the negative controls have shown that the number of false-positive foci does not exceed 15% of the total number of signals. As expected, treatment of cells with a cytotoxic nucleotide hydroxymethyl-deoxyuridine (hmdU) resulted in an increase in the number of detected sSTRIDE-SMUG1 signals. To verify the specificity of the assay, SMUG1 was knocked-down in HAP1 wild-type cells using siRNA and then the level of sSTRIDE-SMUG1 signals was measured in both untreated and hmdU-treated cells. In all tested conditions, SMUG1 expression silencing has resulted in a decrease in sSTRIDE-SMUG1 foci. We believe that the sSTRIDE-SMUG1 assay can prove to be a very useful solution to study DNA repair mechanisms involving aberrant base excision. Additionally, sSTRIDE-SMUG1 measurements may be of the utmost importance in characterization of agents inducing synthetic lethality or trapping BER intermediates. Citation Format: Karolina Stepien, Franek Sierpowski, Zsombor Prucsi, Szymon Koman, Monika Jarosz, Agnieszka Waligorska, Magdalena Kordon-Kiszala, Kamil Solarczyk. Measuring the activity of SMUG1 DNA glycosylase with a novel sSTRIDE-SMUG1 assay abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2066.