Award Title: Identification and nucleotide resolution mapping of alkyl adducts in human DNA
Funder: Cancer Research UK

Abstract: Background Carcinogen induced DNA damage is increasingly implicated as a significant contributor to mutational landscape of human tumours and thereby affects cancer risk. We hypothesise that exposure to endogenous alkylating agents (AAs) arising in situ from N-nitrosatable substrates contributes to this landscape by inducing a spectrum of pro-mutagenic alkyl adducts in DNA. However, the alkyl adductome cannot currently be established due to a lack of appropriate analytical methodology. As AAs are known carcinogens, alkyl adductome characterisation will enable their role in human cancer to finally be defined.   Aims Our overall aim is to characterise the human alkyl adductome at the genomic and nucleotide level. Specifically, we will develop and apply:  1. ion-mobility (IM) coupled to liquid chromatography-mass spectrometry (LC-MS) to characterise O6-alkylguanines (O6-alkGs) and phosphate triesters (PTEs) in human DNA at the genome level. 2. ChIP-exo-based methods to determine the sequence context of O6-alkG distribution.   Methods Oligodeoxyribonucleotides (ODNs) containing O6-alkGs and PTEs with alkyl groups reported to be present in human DNA will be synthesised. These will be incubated with the E. coli ada dual O6-alkG-PTE alkyltransferase (Ada) and transfer of the alkyl groups to the two Ada active sites will be detected and quantified, following protease digestion, by IM-LC-MS. Alkylated active site peptides (ASPs) will be obtained through chemical synthesis and a data repository developed. Human DNA will then be incubated with Ada and the alkylated ASPs analysed enabling the alkyl DNA adductome to be extensively defined at the whole genome level. O6-alkG binding at the nucleotide level will employ ChIP-exonuclease-based methods. Temozolomide-treated DNA will be digested with SauIIIa1 and incubated with Atl1 to isolate O6-alkG containing fragments. These will be further digested with a) lambda exonuclease and processed for next generation sequencing and b) lambda exonuclease and exonuclease I to generate Atl1 footprints for pentanucleotide library hybridation sequencing analyses. To validate these methods, MGMT-expressing T98G glioma cells will be incubated with temozolomide with or without the MGMT inhibitor, lomeguatrib, and the nucleotide distribution of O6-alkGs determined at the gene and nucleotide levels.   How the results of this research will be used  This work will help to provide a more comprehensive picture of the alkyl adductome and will address a fundamental deficit in our understanding of how AA exposure increases mutational and cancer risk. Development of these high throughput biomarker methods will inform cancer risk and prevention strategies at the individual and population levels

Lay Abstract: Background Carcinogen induced DNA damage is increasingly implicated as a significant contributor to mutational landscape of human tumours and thereby affects cancer risk. We hypothesise that exposure to endogenous alkylating agents (AAs) arising in situ from N-nitrosatable substrates contributes to this landscape by inducing a spectrum of pro-mutagenic alkyl adducts in DNA. However, the alkyl adductome cannot currently be established due to a lack of appropriate analytical methodology. As AAs are known carcinogens, alkyl adductome characterisation will enable their role in human cancer to finally be defined.   Aims Our overall aim is to characterise the human alkyl adductome at the genomic and nucleotide level. Specifically, we will develop and apply:  1. ion-mobility (IM) coupled to liquid chromatography-mass spectrometry (LC-MS) to characterise O6-alkylguanines (O6-alkGs) and phosphate triesters (PTEs) in human DNA at the genome level. 2. ChIP-exo-based methods to determine the sequence context of O6-alkG distribution.   Methods Oligodeoxyribonucleotides (ODNs) containing O6-alkGs and PTEs with alkyl groups reported to be present in human DNA will be synthesised. These will be incubated with the E. coli ada dual O6-alkG-PTE alkyltransferase (Ada) and transfer of the alkyl groups to the two Ada active sites will be detected and quantified, following protease digestion, by IM-LC-MS. Alkylated active site peptides (ASPs) will be obtained through chemical synthesis and a data repository developed. Human DNA will then be incubated with Ada and the alkylated ASPs analysed enabling the alkyl DNA adductome to be extensively defined at the whole genome level. O6-alkG binding at the nucleotide level will employ ChIP-exonuclease-based methods. Temozolomide-treated DNA will be digested with SauIIIa1 and incubated with Atl1 to isolate O6-alkG containing fragments. These will be further digested with a) lambda exonuclease and processed for next generation sequencing and b) lambda exonuclease and exonuclease I to generate Atl1 footprints for pentanucleotide library hybridation sequencing analyses. To validate these methods, MGMT-expressing T98G glioma cells will be incubated with temozolomide with or without the MGMT inhibitor, lomeguatrib, and the nucleotide distribution of O6-alkGs determined at the gene and nucleotide levels.   How the results of this research will be used  This work will help to provide a more comprehensive picture of the alkyl adductome and will address a fundamental deficit in our understanding of how AA exposure increases mutational and cancer risk. Development of these high throughput biomarker methods will inform cancer risk and prevention strategies at the individual and population levels

Research Activity Codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health Categories: CANCER AND NEOPLASMS