Characterization of Ribonucleotides Embedded in DNA

Abstract

Ribonucleotides (rNMPs) are the most abundant non-standard nucleotides in genomic DNA. Presence of rNMPs embedded in DNA alters the DNA structure, function and their properties, which ultimately may lead to genomic instability in the form of mutagenesis, replication stress and DNA breaks. Despite abundant evidence of the negative impact of rNMPs in DNA, not much is known about location and identity of rNMPs incorporated in genomic DNA. Here, our aims are to study the genome-wide distribution of rNMPs, and to characterize DNA repair mechanisms responsible for removal of rNMPs and modified rNMPs. To better understand the profile of rNMPs in DNA, we modified our current method, ribose-seq, to generate a robust and effective technique to capture rNMPs incorporated in DNA. Using our modified ribose-seq, in addition to mapping rNMPs in ribonuclease (RNase) H2 null cells at a much higher efficiency, we also determined the rNMP incorporation pattern from wild type DNA of budding yeast S. cerevisiae and S. paradoxus, and fission yeast S. pombe. Additionally, we explored the role of RNase H2 in cleaving modified rNMPs, such as abasic rNMPs. To study if RNase H2 can cleave at abasic rNMPs in DNA, we investigated whether eukaryotic RNase H2 is capable of recognizing abasic rNMPs. Also, we investigated the role of base excision repair (BER) enzymes in cleaving rNMPs and abasic rNMPs. We identity the role of apurinic/apyrimidinic endonuclease 1 (APE1) in cleaving abasic rNMPs, thus revealing a novel function of the BER pathway.