Abstract
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 5′ to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3′-5′ exonuclease activity, which presumably is responsible for cleaning up nonconventional 3′ 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 3′-end nucleotide removal in the 3′-5′ exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5′ dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3′ end of the short oligonucleotide. The impact of the 3′-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3′-5′ exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3′ nucleotide removal by APE1 in the 3′-5′ exonuclease process is the release of the detached nucleotide from the enzyme’s active site.
Highlights
Human apurinic/apyrimidinic endonuclease 1 (APE1), known as a redox factor 1(Ref-1) is a multifunctional enzyme [1]
Our findings revealed that in a 30 -50 exonuclease reaction, removal of the first 30 -terminal nucleotide proceeds significantly faster than the removal of subsequent nucleotides located at the positions from the 30 end
This finding indicates that catalysis during the first enzymatic turnover is more rapid than in the subsequent enzymatic cycles, which supports the appearance of the rate-limiting step after catalytic hydrolysis of the phosphodiester bond
Summary
Human apurinic/apyrimidinic endonuclease 1 (APE1), known as a redox factor 1(Ref-1) is a multifunctional enzyme [1]. The currently available structural data mainly represent different variants of the free enzyme [17,18,19] and its complexes with nicked or abasic DNA [20,21,22]. Analysis of these data has shown that, for endonuclease activity, specific contacts in the complex APE1·DNA are formed, which result in the change of DNA conformation including eversion of an AP-site from the double helix. It has been shown that amino acid residues of APE1 interact mainly with only one DNA chain, which contains an AP-site by forming
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