Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity

A. A. Kuznetsova, A. A. Gavrilova, D. S. Novopashina, O. S. Fedorova, N. A. Kuznetsov

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1 Citation (Scopus)

Abstract

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine–purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine–purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

Translated title of the contributionМутационный и кинетический анализ эндорибонуклеазной активности АРЕ1
Original languageEnglish
Article number5
Pages (from-to)211-224
Number of pages14
JournalMolecular Biology
Volume55
Issue number2
DOIs
Publication statusPublished - Mar 2021

Keywords

  • active site
  • APE1
  • endoribonuclease activity
  • human apurine/apyrimidine endonuclease
  • RNA substrates
  • site-directed mutagenesis

OECD FOS+WOS

  • 1.06 BIOLOGICAL SCIENCES

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