Chemical Modifications in Regulatory Regions of DNA and RNA

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Project Summary/Abstract Both the DNA genome and the RNA transcriptome are punctuated with chemical modifications to the four bases, and these modifications can either be added to individual nucleotides by specific enzymes or installed by cellular chemistry, such as oxidative stress. This program seeks to understand where these base modifications occur by developing new chemical methods of sequencing for modifications as well as examining the biological outcomes of such modifications through biochemical studies of the proteins that write, read and erase the chemical modifications. Previous NIH support of this program led to the discovery of oxidized guanine nucleotides as epigenetic-like modifications in G-quadruplexes of gene promoters as well as sequencing methods to characterize oxidation in the genome and base modifications in the transcriptome. Future work will study the key DNA repair proteins, apurinic/apyrimidinic endonuclease-1 (APE1), oxidized guanine glycosylase 1 (OGG1), endonuclease VIII-like protein 1 (NEIL1) and Cockayne syndrome B (CSB) protein by biophysical methods to understand the mechanism of gene regulation. For both DNA and RNA, nanopore sequencing methods will be advanced for detection of oxidized bases as well as the two most common base modifications in RNA, pseudouridine and N6-methyladenosine. These unusual bases are found to be more abundant in cells undergoing oxidative or inflammatory stress and viral infection.