Multiple studies have identified mechanisms by which ribonucleotides can be added to, removed from, or generated from DNA. Primarily, ribonucleotides are incorporated into DNA during the DNA synthesis process. To initiate DNA replication, short RNA primers synthesized on complementary DNA to allow for the subsequent binding and replication action of the primary replication enzymes, DNA polymerases. Typically, these primers are then removed by nuclease enzymes, RNases H or Flap Structure-specific Endonuclease 1 (FEN1). As such, there is a fairly substantial volume of transient rNMPs that are present in DNA in the form of RNA primers and then later removed. After DNA replication is initiated, rNMPs can be fully incorporated into DNA by DNA polymerases and this represents the major mechanism of ribonucleotide incorporation in DNA with over 1 ribonucleotide per 1,000 deoxyribonucleotides incorporated. The embedded rNMPs are often targeted for removal by the Ribonucleotide Excision Repair (RER) mechanism. However, if RER is not working properly, this can become a more persistent source of embedded rNMPs in DNA. Another mechanism by which rNMPs may arise in DNA is through oxidative stress, which alters the deoxyribose unit in DNA to a ribose. While the RER pathway is the most efficient process for removing rNMPs from DNA, ribonucleotides can also be removed through the 3’-5’ exonuclease activity that is orchestrated by the proofreading activities of DNA polymerase during DNA replication. Additional rNMP removal methods include incisions that are mediated by the isomerase enzyme, topoisomerase I.
