RESUMEN
In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.
Asunto(s)
ADN/genética , Genoma/genética , Análisis de Secuencia de ADN/métodos , Aldehídos/química , Secuencia de Bases , ADN/química , Daño del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Leishmania major/genética , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Timina/química , Uracil-ADN Glicosidasa/químicaRESUMEN
While some DNA base modifications such as 5-methylcytosine have been known and studied for decades, recent discoveries of a number of other modified bases have stimulated research to understand their origin and function. Chemistry-based methods for their detection and analysis have proven to be important for advancing the field. Here, we feature a selection of methods that have helped advance the field, along with some key advances in the understanding of how the chemistry of modified bases affects biological functions. We also discuss fundamental questions in the field that remain unanswered.
