TY - JOUR

T1 - Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity

AU - Kuznetsova, A. A.

AU - Gavrilova, A. A.

AU - Novopashina, D. S.

AU - Fedorova, O. S.

AU - Kuznetsov, N. A.

N1 - Funding Information: The experimental work was supported by the Russian Science Foundation (project no. 19-74-10034) and partially supported by budgetary funding to ensure routine maintenance on the equipment No. АААА-А17-117020210022-4. Publisher Copyright: © 2021, Pleiades Publishing, Inc.

PY - 2021/3

Y1 - 2021/3

N2 - 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.

AB - 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.

KW - active site

KW - APE1

KW - endoribonuclease activity

KW - human apurine/apyrimidine endonuclease

KW - RNA substrates

KW - site-directed mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=85125578323&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/7e80fb0f-82c3-343e-8bd5-18dd6d3b715b/

U2 - 10.1134/S0026893321020102

DO - 10.1134/S0026893321020102

M3 - Article

C2 - 33948042

AN - SCOPUS:85125578323

VL - 55

SP - 211

EP - 224

JO - Molecular Biology

JF - Molecular Biology

SN - 0026-8933

IS - 2

M1 - 5

ER -