Coordination between human DNA polymerase ß and apurinic/apyrimidinic endonuclease 1 in the course of DNA repair.

Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polß in BER through studying an impact of APE1 on Polß-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polß's catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3' blocking groups and corrected Polß-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polß not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polß during the BER process.

The Impact of Human DNA Glycosylases on the Activity of DNA Polymerase ß toward Various Base Excision Repair Intermediates.

Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase ß, and DNA ligase. Coordination of BER is implemented by multiple protein-protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polß's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polß efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5'-dRP-mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polß's activity toward the model DNA intermediates.

Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase ß on the APE1-DNA interaction.

The base excision repair (BER) pathway involves sequential action of DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases to incise damaged DNA and prepare DNA termini for incorporation of a correct nucleotide by DNA polymerases. It has been suggested that the enzymatic steps in BER include recognition of a product-enzyme complex by the next enzyme in the pathway, resulting in the "passing-the-baton" model of transfer of DNA intermediates between enzymes. To verify this model, in this work, we aimed to create a suitable experimental system. We prepared APE1 site-specifically labeled with a fluorescent reporter that is sensitive to stages of APE1-DNA binding, of formation of the catalytic complex, and of subsequent dissociation of the enzyme-product complex. Interactions of the labeled APE1 with various model DNA substrates (containing an abasic site) of varied lengths revealed that the enzyme remains mostly in complex with the DNA product. By means of the fluorescently labeled APE1 in combination with a stopped-flow fluorescence assay, it was found that Polß stimulates both i) APE1 binding to an abasic-site-containing DNA duplex with the formation of a catalytically competent complex and ii) the dissociation of APE1 from its product. These findings confirm DNA-mediated coordination of APE1 and Polß activities and suggest that Polß is the key trigger of the DNA transfer between the enzymes participating in initial steps of BER.

Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease.

Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5' side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3'-5'-exonuclease, 3'-phosphodiesterase, and 3'-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre-steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding-site residue, Arg181, was analyzed too; it changed its conformation when enzyme-substrate and enzyme-product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction.

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis.

In the repair of the damage to bases, human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key participant via the DNA base excision repair pathway. APE1 cleaves AP sites in DNA, which are potentially cytotoxic and highly mutagenic if left unrepaired. According to existing structural data, this enzyme's active site contains many polar amino acid residues, which form extensive contacts with a DNA substrate. A few alternative catalytic mechanisms of the phosphodiester bond hydrolysis by APE1 have been reported. Here, the kinetics of conformational changes of the enzyme and of DNA substrate molecules were studied during the recognition and cleavage of the abasic site in the pH range from 5.5 to 9.0 using stopped-flow fluorescence techniques. The activity of APE1 increased with an increase in pH because of acceleration of the rates of catalytic complex formation and of the catalytic reaction. Molecular dynamics simulation uncovered a significant increase in the pKa of His-309 located in the active site of the enzyme. This finding revealed that the observed enhancement of enzymatic activity with pH could be associated with deprotonation of not only Tyr-171 but also His-309. The obtained data allowed us to hypothesize that the ionized state of these residues could be a molecular switch between the alternative catalytic mechanisms, which involve different functionalities of these residues throughout the reaction.