Investigation/Characterization of base modifications in mammalian DNA

Abstract

Deoxyribonucleic acid (DNA) is the macromolecule carrying the majority of heritable information. Beyond the sequence of the four canonical nucleotides, chemical modifications can add another layer of information. In mammalian DNA, so far five enzymatically introduced modifications have been described. DNA modifications can play major biological roles in transcription regulation, chromatin structure and accessibility. In this study, I explored unknown, epigenetic DNA modifications and investigated the regulation and biological functions of N6 methylated deoxyadenosine (m6dA) and deoxyinosine (dI) in mammalian DNA. In the first part of my thesis, to identify and characterize novel epigenetic DNA modifications, I used a mass spectrometry-based screening method. However, a gain-of-function screen with potential DNA modifying enzymes and a screen for sulfur-containing DNA modifications did not reveal any new base modifications in human DNA. In the second part of my thesis, applying the screen approach on DNA:RNA hybrid regions (R-loops) in the mammalian genome showed that deoxyinosine (dI), a deamination product of deoxyadenosine (dA), is enriched within R loops. Though dI is commonly regarded as DNA damage, its specific enrichment within R-loops suggests a biological significance of this modification. Consistently, my results indicate that adenosine deaminase acting on RNA 1 (ADAR1), an enzyme well characterized for its RNA editing activity, promoted dI formation in DNA:RNA hybrids. Although this study does not provide ultimate proof on the biological consequences, it raises the possibility that ADAR-mediated editing of DNA in DNA:RNA hybrids affects R-loop stability and clearance. In the third part of my thesis, I contributed to investigating the role and origin of N6-methyl deoxyadenosine (m6dA) in mammalian DNA, which remains controversial.