Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage

Yu Xu,Akanksha Manghrani,Bei Liu,Honglue Shi,Uyen Pham,Amy Liu,Hashim M Al-Hashimi

doi:10.1074/jbc.ra120.014530

Abstract

As the Watson-Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson-Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

Highlights

The Watson-Crick faces of nucleobases are tucked in the interior of the DNA double helix where they are largely inaccessible to solvent, shielded by Watson-Crick hydrogen bonding, and protected from endogenous and environmental agents that may cause various deleterious forms of alkylation damage[1,2,3]
Our results reveal a mechanism for damaging Watson-Crick faces of DNA via A-T Hoogsteen bps without the need for melting dsDNA, and establish the utility of dimethyl sulfate (DMS) probing in characterizing A(syn)-T Hoogsteen base pairs in addition to unpaired adenines in dsDNA in vitro
We developed an antibody-based rescue-coupled dot-blot assay to detect and quantify m1A following treatment of DNA oligonucleotides with DMS

Summary

Introduction

The Watson-Crick faces of nucleobases are tucked in the interior of the DNA double helix where they are largely inaccessible to solvent, shielded by Watson-Crick hydrogen bonding, and protected from endogenous and environmental agents that may cause various deleterious forms of alkylation damage[1,2,3]. It is generally accepted that alkylation damage to the Watson-Crick faces of nucleobases by endogenous and environmental agents as well as anti-cancer therapies [5,6,12] occurs primarily during replication and transcription, when the DNA is transiently single-stranded [4]. In prokaryotes, the activities of enzymes that repair alkylation damage have been linked to the process of DNA replication [4,15]

Methods

Results

Conclusion