APE1 Cleavage of Abasic Ribonucleotides Embedded in DNA Nicole M. Hoitsma and Bret D. Freudenthal, University of Kansas Medical Center Ribonucleotides (rNTPs) are incorporated into the genome at a rate of ~3 million times per cell division, making rNTPs the most common non-standard nucleotide found in the human genome. Typically, mis-inserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage 5’ of the rNTP. However, rNTPs embedded in DNA are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is important for the initial processing of rAPs embedded in DNA. APE1 is a well characterized AP-endonuclease that cleaves 5’ of abasic deoxynucleotides. However, its ability to cleave at rAPs remains poorly understood. Here, we utilize X-ray crystallography and enzyme kinetics to provide molecular insight into rAP processing by APE1. Using X-ray crystallography, we identified novel contacts between rAP and the APE1 active site that are unique from other APE1:DNA complexes. These include novel interactions with the 5’ phosphate of the rAP that suggest APE1 may have altered metal dependence when facilitating catalysis on rAP substrates. In addition, we identified APE1 active site residues that are important for accommodating the 2’ OH of the rAP. Finally, enzyme kinetics were used to determine the pre-steady-state kinetic rates of wild-type APE1 cleavage on DNA substrates containing rAP. Together, this work provides molecular level insights into rAP processing by APE1 and advances the comprehensive understanding of the diverse functions of APE1.