Abstract 5780: Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1
Authors: Yaakub Hanum , Howell Anthony , Margison Geoffrey , Povey Andrew 
Published: September 2020

Abstract
Humans are widely exposed to a wide variety of DNA alkylating agents (AAs) either directly, for example via cigarette smoke or indirectly via their formation in the gastrointestinal tract. Certain AAs, such as the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) are known human carcinogens but the role that other AAs may play in the aetiology of human cancers is still uncertain due to difficulties in assessing human exposure to these agents. The aim of this work was therefore to develop a screening method to assess exposure to AAs by measuring the formation of O6-alkylguanines (O6-AlkGs) in DNA and applying it to the analysis of human DNA samples. To do this we took advantage of the S. pombe alkyltransferase-like protein (Atl1), which binds to all O6-AlkGs so far studied, to develop a slot blot assay. For this, sonicated heat denatured DNA was bound to Hybond-N+ membranes and probed with horseradish peroxidase-conjugated maltose-binding protein-Atl1 fusion protein. The binding was quantitated via peroxidase-mediated enhanced chemiluminescence using TMZ-methylated calf thymus (CT) DNA standards previously calibrated using a tritium-based competition assay. Using 1 μg DNA, the limit of quantitation and detectability were approximately 1.0 and 0.5 fmoles O6-MeG/μg DNA, respectively. Prior treatment of the TMZ-CT DNA standards with the DNA repair protein, O6-methylguanine DNA methyltransferase, reduced the detectable signal to background levels, confirming that the assay detected O6-methylguanine (O6-MeG) in TMZ-CT DNA. Importantly, CT DNA reacted in vitro with the alkylating agents N-ethyl-N-nitrosourea, N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, N-benzyl-N-nitrosourea, N-allyl-N-nitrosourea and potassium diazoacetate all produced strong signals in the assay demonstrating its ability to detect a range of different O6-AlkGs. In addition, a variety of human DNA samples have been analysed in pilot studies: of 14 human placental DNA samples, 8 had O6-AlkG levels in the range of 1.05-1.71 fmoles/μg DNA, 3 had trace levels and in 3 samples there was no detectable signal. Of 5 human breast tumour DNA samples, 4 had O6-AlkGs levels in the range of 1.07-1.30 fmoles/µg DNA and 1 had trace levels. Two DNA samples from normal human breast tissue had no detectable O6-AlkGs but DNA from the tumours of the same patients had levels &gt;1 fmoles/μg DNA. Of 6 DNA samples from whole blood 2 had detectable levels of O6-AlkGs (1.23 & 1.56 fmoles/μg DNA), 3 had trace amounts and 1 had no detectable level. These data thus show the potential usefulness of this slot blot assay as a screening tool to detect and quantify total O6-alkGs adducts in human DNA samples.
Citation Format: Hanum Yaakub, Anthony Howell, Geoffrey Margison, Andrew Povey. Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5780.