SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polß Polymerase Activity.

In the cell, DNA polymerase ß (Polß) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polß can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polß variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polß functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions-G274R and R333W-led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate-binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage.

The Impact of SNP-Induced Amino Acid Substitutions L19P and G66R in the dRP-Lyase Domain of Human DNA Polymerase ß on Enzyme Activities.

Base excision repair (BER), which involves the sequential activity of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases, is one of the enzymatic systems that preserve the integrity of the genome. Normal BER is effective, but due to single-nucleotide polymorphisms (SNPs), the enzymes themselves-whose main function is to identify and eliminate damaged bases-can undergo amino acid changes. One of the enzymes in BER is DNA polymerase ß (Polß), whose function is to fill gaps in DNA. SNPs can significantly affect the catalytic activity of an enzyme by causing an amino acid substitution. In this work, pre-steady-state kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring variants of Polß that have the substitutions L19P and G66R in the dRP-lyase domain. Despite the substantial distance between the dRP-lyase domain and the nucleotidyltransferase active site, it was found that the capacity to form a complex with DNA and with an incoming dNTP is significantly altered by these substitutions. Therefore, the lower activity of the tested polymorphic variants may be associated with a greater number of unrepaired DNA lesions.

The Activity of Natural Polymorphic Variants of Human DNA Polymerase ß Having an Amino Acid Substitution in the Transferase Domain.

To maintain the integrity of the genome, there is a set of enzymatic systems, one of which is base excision repair (BER), which includes sequential action of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases. Normally, BER works efficiently, but the enzymes themselves (whose primary function is the recognition and removal of damaged bases) are subject to amino acid substitutions owing to natural single-nucleotide polymorphisms (SNPs). One of the enzymes in BER is DNA polymerase ß (Polß), whose function is to fill gaps in DNA with complementary dNMPs. It is known that many SNPs can cause an amino acid substitution in this enzyme and a significant decrease in the enzymatic activity. In this study, the activity of four natural variants of Polß, containing substitution E154A, G189D, M236T, or R254I in the transferase domain, was analyzed using molecular dynamics simulations and pre-steady-state kinetic analyses. It was shown that all tested substitutions lead to a significant reduction in the ability to form a complex with DNA and with incoming dNTP. The G189D substitution also diminished Polß catalytic activity. Thus, a decrease in the activity of studied mutant forms may be associated with an increased risk of damage to the genome.

Human Polß Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis.

DNA polymerase ß (Polß) expression is essential for the cell's response to DNA damage that occurs during natural cellular processes. Polß is considered the main reparative DNA polymerase, whose role is to fill the DNA gaps arising in the base excision repair pathway. Mutations in Polß can lead to cancer, neurodegenerative diseases, or premature aging. Many single-nucleotide polymorphisms have been identified in the POLB gene, but the consequences of these polymorphisms are not always clear. It is known that some polymorphic variants in the Polß sequence reduce the efficiency of DNA repair, thereby raising the frequency of mutations in the genome. In the current work, we studied two polymorphic variants (G118V and R149I separately) of human Polß that affect its DNA-binding region. It was found that each amino acid substitution alters Polß's affinity for gapped DNA. Each polymorphic variant also weakens its binding affinity for dATP. The G118V variant was found to greatly affect Polß's ability to fill gapped DNA and slowed the catalytic rate as compared to the wild-type enzyme. Thus, these polymorphic variants seem to decrease the ability of Polß to maintain base excision repair efficiency.

The Role of Natural Polymorphic Variants of DNA Polymerase ß in DNA Repair.

DNA polymerase ß (Polß) is considered the main repair DNA polymerase involved in the base excision repair (BER) pathway, which plays an important part in the repair of damaged DNA bases usually resulting from alkylation or oxidation. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that protein-protein interactions of Polß with enzymes from the BER pathway increase the efficiency of damaged base repair in DNA. However natural single-nucleotide polymorphisms can lead to a substitution of functionally significant amino acid residues and therefore affect the catalytic activity of the enzyme and the accuracy of Polß action. Up-to-date databases contain information about more than 8000 SNPs in the gene of Polß. This review summarizes data on the in silico prediction of the effects of Polß SNPs on DNA repair efficacy; available data on cancers associated with SNPs of Polß; and experimentally tested variants of Polß. Analysis of the literature indicates that amino acid substitutions could be important for the maintenance of the native structure of Polß and contacts with DNA; others affect the catalytic activity of the enzyme or play a part in the precise and correct attachment of the required nucleotide triphosphate. Moreover, the amino acid substitutions in Polß can disturb interactions with enzymes involved in BER, while the enzymatic activity of the polymorphic variant may not differ significantly from that of the wild-type enzyme. Therefore, investigation regarding the effect of Polß natural variants occurring in the human population on enzymatic activity and protein-protein interactions is an urgent scientific task.

Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein-protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polß; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer-based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein-protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein-protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein-protein interactions in the coordination of the repair pathway.

Conformational Dynamics of Damage Processing by Human DNA Glycosylase NEIL1.

Endonuclease VIII-like protein 1 (NEIL1) is a DNA repair enzyme found in higher eukaryotes, including humans. It belongs to the helix-two turn-helix (H2TH) structural superfamily together with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei), and removes a variety of oxidized purine and pyrimidine bases from DNA. Structural, modeling and kinetic studies have established that the bacterial H2TH superfamily enzymes proceed through several conformational intermediates while recognizing and removing their cognate lesions. Here we apply stopped-flow kinetics with detection of intrinsic Trp fluorescence and Förster resonance energy transfer fluorescence to follow the conformational dynamics of human NEIL1 and DNA when the enzyme interacts with undamaged DNA, or DNA containing cleavable or non-cleavable abasic sites, or dihydrouracil lesions. NEIL1 processed a natural abasic site and a damaged base in DNA equally well but showed an additional fluorescently discernible step when DHU was present, likely reflecting additional rearrangements during base eversion into the enzyme's active site. With undamaged DNA and DNA containing a non-cleavable abasic site analog, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate, NEIL1 was diverted to a non-productive DNA conformation early in the reaction. Our results support the view of NEIL1 as an enzyme that actively destabilizes damaged DNA and uses multiple checkpoints along the reaction coordinate to drive substrate lesions into the active site while rejecting normal bases and non-substrate lesions.

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms.

Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5-37 °C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van't Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme-substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme-DNA complex formation.

The role of the N-terminal domain of human apurinic/apyrimidinic endonuclease 1, APE1, in DNA glycosylase stimulation.

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3'-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl-N-purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1-DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme-substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate. SIGNIFICANCE STATEMENT: The major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair.

Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII.

Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and ß, δ-elimination of 3'- and 5'-phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5'-phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does. Therefore, most likely, Leu70 plays the role of a DNA lesion "sensor", which is used by Endo VIII for recognition of a DNA damage site.

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III.

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.