Lys98 Substitution in Human AP Endonuclease 1 Affects the Kinetic Mechanism of Enzyme Action in Base Excision and Nucleotide Incision Repair Pathways

Abstract

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5′-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates.

Unveiling the non-repair face of the Base Excision Repair pathway in RNA processing: A missing link between DNA repair and gene expression?

“…In the last decade, Nucleotide Incision Repair (NIR) pathway has been described as a new function of APE1 which works as back up of BER pathway ensuring a correct removal of damaged bases as a result of oxidative stress [20][21][22][23][24][25]. NIR activity by APE1 consists of an incision at the 5′ next to a oxidatively damaged base in a DNA glycosylase-independent manner, providing a proper 3′-OH group for further processing [20].…”

“…Interestingly, the N-terminal domain of APE1 deputed to modulate proteinprotein interaction and indispensable to redox activity but not APendonuclease activity [38], is contrarily essential for the NIR activity; indeed the lack of the first 33 N-terminal aminoacids of the protein produces a 20-fold-decrease of APE1 NIR activity [20]. Moreover, Timofeyeva et al demonstrated that Lysine in position 98 contributes significantly in the 5′-phospodiester bond hydrolysis of DNA substrate, but not in the dissociation of the enzyme-product complex [21]. Interestingly, the substitution of this amino acid influences the APE1 NIR activity more than BER, demonstrating that the APE1 active site involved in NIR and BER pathways is the same, but different conformational requirements are responsible for APE1 NIR or BER activities [21].…”

“…Moreover, Timofeyeva et al demonstrated that Lysine in position 98 contributes significantly in the 5′-phospodiester bond hydrolysis of DNA substrate, but not in the dissociation of the enzyme-product complex [21]. Interestingly, the substitution of this amino acid influences the APE1 NIR activity more than BER, demonstrating that the APE1 active site involved in NIR and BER pathways is the same, but different conformational requirements are responsible for APE1 NIR or BER activities [21].…”

Types and effects of protein variations

“…The enzyme-substrate complex undergoes induced fit upon binding to lesion. Substitution K98A in APE1 has an "effect on induced fit" preventing it and decreasing the stability of enzyme-substrate complex (Timofeyeva et al 2011). …”

DNA damage and base excision repair : an important role during zebrafish embryogenesis

“…This lesion would have been generated by the 5' incision repair activity of APEX1 (Timofeyeva et al 2011). Methylation of both C residues on both strands in the CpG islet, e.g.…”