TY - JOUR

T1 - Kinetic features of 30-50 exonuclease activity of human AP-endonuclease ape1

AU - Kuznetsova, Alexandra A.

AU - Fedorova, Olga S.

AU - Kuznetsov, Nikita A.

N1 - Publisher Copyright: © 2018 by the authors.

PY - 2018/8/21

Y1 - 2018/8/21

N2 - Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 50 to the damage. In addition to the AP-endonuclease activity, APE1 possesses 30-50 exonuclease activity, which presumably is responsible for cleaning up nonconventional 30 ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 30-end nucleotide removal in the 30-50 exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 50 dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 30 end of the short oligonucleotide. The impact of the 30-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 30-50 exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 30 nucleotide removal by APE1 in the 30-50 exonuclease process is the release of the detached nucleotide from the enzyme’s active site.

AB - Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 50 to the damage. In addition to the AP-endonuclease activity, APE1 possesses 30-50 exonuclease activity, which presumably is responsible for cleaning up nonconventional 30 ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 30-end nucleotide removal in the 30-50 exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 50 dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 30 end of the short oligonucleotide. The impact of the 30-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 30-50 exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 30 nucleotide removal by APE1 in the 30-50 exonuclease process is the release of the detached nucleotide from the enzyme’s active site.

KW - AP-endonuclease

KW - DNA repair

KW - Exonuclease activity

KW - Pre-steady-state kinetics

KW - Humans

KW - Substrate Specificity

KW - DNA/chemistry

KW - DNA Repair

KW - Catalysis

KW - Enzyme Activation

KW - Kinetics

KW - Exonucleases/chemistry

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry

KW - RECOGNITION

KW - INCISION REPAIR PATHWAY

KW - pre-steady-state kinetics

KW - SITE

KW - ABASIC DNA

KW - exonuclease activity

KW - CONFORMATIONAL DYNAMICS

KW - FLUORESCENCE

KW - DIVALENT METAL-IONS

KW - BASE-EXCISION

KW - DNA-REPAIR

KW - HUMAN APURINIC/APYRIMIDINIC ENDONUCLEASE

UR - http://www.scopus.com/inward/record.url?scp=85052817828&partnerID=8YFLogxK

U2 - 10.3390/molecules23092101

DO - 10.3390/molecules23092101

M3 - Article

C2 - 30134601

AN - SCOPUS:85052817828

VL - 23

JO - Molecules

JF - Molecules

SN - 1420-3049

IS - 9

M1 - 2101

ER -