Abasic (AP) sites are one of the most abundant DNA modifications produced at an estimated rate of 10,000 AP sites per cell per day in mammalian cells. Major sources of AP sites include base excision repair, depurination/depyrimidination, and enzymatic processing of DNA epigenetic marks. AP sites exist in an equilibrating mixture of the ring-closed hemiacetyl form and the ring-open aldehyde form. Considering that 5-formylcytosine and 5-formyluracil also contain an aldehyde group and the high abundance of these modifications under certain biological conditions, highly selective and efficient detection AP sites in the presence of other aldehydic lesions remains challenging. Here, we developed a two chemicals-assisted high-resolution mapping method for AP sites at single-nucleotide resolution. Our method feature an azido modified hydrazide group derivative to selectively label AP sites and a hydroxylamine functionized cytosine analogue to provide signal readout. For proof-of-concept, we mapped AP sites in mitochondrial DNA isolated from human HeLa cells and considered our results in the context of mitochondrial DNA packaging. Sequencing AP sites paves the way for a deeper understanding of their biological functions in mitochondria.

Single-nucleotide resolution mapping of abasic sites in mitochondrial DNA

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ACS Fall 2022

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