Substrate specificity and activity of extremotolerant tardigrade AP-endonuclease 1.

Abstract

Tardigrades are microinvertebrates known for their extreme resilience to ionizing radiation, desiccation, and other environmental stresses. This tolerance is partly attributed to efficient DNA repair mechanisms. Here, we biochemically characterized the apurinic/apyrimidinic endonuclease 1 from Hypsibius exemplaris (hexAPE1), a multifunctional enzyme in the base excision repair (BER) pathway. Sequence analysis confirmed that hexAPE1 belongs to the ExoIII family and shares conserved catalytic residues with human APE1. Using synthetic DNA and RNA substrates, we demonstrated that hexAPE1 exhibits three distinct activities: AP-endonuclease, nucleotide incision repair (NIR), and 3'-5'-exonuclease. The enzyme efficiently cleaved abasic site in DNA duplex and a DNA/RNA hybrid. Notably, hexAPE1 showed a strong preference for the RNA strand in DNA/RNA hybrid substrates. The 3'-5'-exonuclease degradation of the RNA strand in DNA/RNA hybrids was 30-fold faster than that of the DNA strand in DNA duplexes. Moreover, incision of an abasic site within the RNA strand of a hybrid occurred with a rate constant, significantly exceeding that for DNA duplexes. NIR activity was confirmed against damaged nucleotides, including 5,6-dihydrouridine, α-anomer of adenosine, and 2'-deoxyuridine. The ability of hexAPE1 to process both DNA and RNA lesions, particularly within DNA/RNA hybrids, allows to suggest involvement of AP-endonuclease in the cellular quality control of RNA and, probably, damaged RNA clearance process, as well as in the prevention of R-loop-induced genomic instability.