122 Kinetics of apurinic/apyrimidinic endonuclease 1 (APE1) on an authentic AP site or an AP site analog

Our genomic DNA is endlessly exposed to a wide variety of exogenous and endogenous DNA-damaging agents. One of the most abundant DNA lesions is an apurinic/apyrimidinic (AP) site, which in vivo, can form spontaneously or through various cellular pathways, including the repair activity of DNA glycosylase enzymes (Wilson & Barsky, 2001). Persistence of these AP sites is both highly mutagenic and cytotoxic to the cell (Loeb & Preston, 1986). AP endonuclease 1 (APE1), an Mg²⁺ dependent enzyme, is the major human endonuclease responsible for incising the DNA backbone at AP sites. Repair to canonical duplex DNA is then completed by DNA polymerase and DNA ligase. Recently, APE1, in conjunction with delivery of DNA-damaging agents, has become a target for chemotherapeutic research with the aim to inhibit APE1 activity (Fishel & Kelley, 2007). Therefore, an understanding of APE1 activity and its molecular mechanism is essential. In vitro, the authentic AP site is highly unstable and can undergo β-elimination, leading to a strand break (Strauss, Beard, Patterson & Wilson, 1997). Due to the fragility of the AP site, stable AP site analogs, such as the reduced AP site or tetrahydrofuran (THF) site, are typically used to study APE1 (Maher & Bloom, 2007; Strauss, Beard, Patterson & Wilson, 1997). In this work, we have performed the first comprehensive kinetic study of APE1 acting on the authentic AP site as well the reduced AP site and THF AP site analog. Transient-state kinetic experiments reveal that the strand incision chemistry step is fast, upwards of ～700?s^?1 for all substrates, making APE1 one of the fastest DNA repair enzymes. Steady-state kinetic experiments reveal for each substrate, a slow, post chemistry step limits the steady-state rate. The steady-state rate for APE1 acting on authentic AP and AP-Red substrates is highly dependent on Mg²⁺ concentration, while the steady-state rate for THF site was not dependent on Mg²⁺ concentration. This comprehensive kinetic analysis reveal differences and similarities in the way APE1 processes the authentic AP site compared to AP site analogs. Furthermore, these differences require consideration when choosing AP site analogs to study APE1.     

Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision

The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1.     

Conformational dynamics of abasic DNA upon interactions with AP endonuclease 1 revealed by stopped-flow fluorescence analysis

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.     

Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease

Human 8-oxoguanine-DNA glycosylase (OGG1) is the main human base excision protein that removes a mutagenic lesion 8-oxoguanine (8-oxoG) from DNA. Since OGG1 has DNA glycosylase and weak abasic site (AP) lyase activities and is characterized by slow product release, turnover of the enzyme acting alone is low. Recently it was shown that human AP endonuclease (APE1) enhances the activity of OGG1. This enhancement was proposed to be passive, resulting from APE1 binding to or cleavage of AP sites after OGG1 dissociation. Here we present evidence that APE1 could actively displace OGG1 from its product, directly increasing the turnover of OGG1. We have observed that APE1 forms an electrophoretically detectable complex with OGG1 cross-linked to DNA by sodium borohydride. Using oligonucleotide substrates with a single 8-oxoG residue located in their 5'-terminal, central or 3'-terminal part, we have demonstrated that OGG1 activity does not increase only for the first of these three substrates, indicating that APE1 interacts with the DNA stretch 5' to the bound OGG1 molecule. In kinetic experiments, APE1 enhanced the product release constant but not the rate constant of base excision by OGG1. Moreover, OGG1 bound to a tetrahydrofuran analog of an abasic site stimulated the activity of APE1 on this substrate. Using a concatemeric DNA substrate, we have shown that APE1 likely displaces OGG1 in a processive mode, with OGG1 remaining on DNA but sliding away in search for a new lesion. Altogether, our data support a model in which APE1 specifically recognizes an OGG1/DNA complex, distorts a stretch of DNA 5' to the OGG1 molecule, and actively displaces the glycosylase from the lesion.     

The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: Stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue

Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding.     

Multifunctional human apurinic/apyrimidinic endonuclease 1: the role of additional functions

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is multifunctional enzyme. APEI is involved in the DNA base excision repair process (BER). APE1 participates in BER by cleaving the DNA adjacent to the 5' side of an AP site to produce a hydroxyl group at the 3' terminus of an unmodified nucleotide upstream of the nick and a 5' deoxyribose phosphate moiety downstream. In addition to its AP-endonucleolytic function, APE1 possesses 3' phosphodiesterase, 3'-5' exonuclease and 3' phosphatase activities. Independently of being characterized as DNA repair protein, APE1 was identified as redox-factor (Ref-1). Our own and literature data on the role of APE1 additional functions in cell metabolism and on interactions of APE1 with DNA and other proteins that participate in BER are analyzed in this review.     

8-OxoA inhibits the incision of an AP site by the DNA glycosylases Fpg, Nth and the AP endonuclease HAP1

Ionizing radiation induces clustered DNA damage sites, whereby two or more individual DNA lesions are formed within one or two helical turns of DNA by a single radiation track. A subset of DNA clustered damage sites exist in which the lesions are located in tandem on the same DNA strand. Recent studies have established that two closely opposed lesions impair the repair machinery of the cell, but few studies have investigated the processing of tandem lesions. In this study, synthetic double-stranded oligonucleotides were synthesized to contain 8-oxoA and an AP site in tandem, separated by up to four bases in either a 5' or 3' orientation. The influence 8-oxoA has on the incision of the AP site by the E. coli glycosylases Fpg and Nth protein and the human AP endonuclease HAP1 was assessed. 8-OxoA has little or no effect on the efficiency of incision of the AP site by Nth protein; however, the efficiency of incision of the AP site by Fpg protein is reduced in the presence of 8-oxoA even up to a four-base separation in both the 5' and 3' orientations. 8-OxoA influences the efficiency of HAP1 incision of the AP site only when it is 3' to the AP site and separated by up to two bases. This study demonstrates that the initial stages of base excision repair can be impaired by the presence of a second base lesion in proximity to an AP site on the same DNA strand. This impairment could have biological consequences, such as mutation induction, if the AP site is present at replication.     

AP endonuclease 1 has no biologically significant 3(')-->5(')-exonuclease activity

The 3(')-->5(')-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3(')-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3(')-->5(')-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3(')-->5(')-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair.     

Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3' ends justify the means

DNA damage occurs unceasingly in all cells. Spontaneous DNA base loss, as well as the removal of damaged DNA bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinic/apyrimidinic (AP) sites. AP sites are the central intermediate in DNA base excision repair (BER) and must be processed by 5' AP endonucleases. These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5' of, AP sites to create a free 3'-OH end for DNA polymerase repair synthesis. In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these organisms and are homologous to the Escherichia coli enzymes Exonuclease III (Exo III) and Endonuclease IV (Endo IV), respectively. These enzymes represent both of the conserved 5' AP endonuclease enzyme families that exist in biology. Crystal structures of APE1 and Endo IV, both bound to AP site-containing DNA reveal how abasic sites are recognized and the DNA phosphodiester backbone cleaved by these two structurally unrelated enzymes with distinct chemical mechanisms. Both enzymes orient the AP-DNA via positively charged complementary surfaces and insert loops into the DNA base stack, bending and kinking the DNA to promote flipping of the AP site into a sequestered enzyme pocket that excludes undamaged nucleotides. Each enzyme-DNA complex exhibits distinctly different DNA conformations, which may impact upon the biological functions of each enzyme within BER signal-transduction pathways.     

AP endonuclease 1 as a key enzyme in repair of apurinic/apyrimidinic sites

Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5′-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3′-hydroxyl group and a 5′-deoxyribose phosphate moiety. APE1 exhibits 3′-phosphodiesterase, 3′-5′-exonuclease, and 3-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system.     

Bcl2 inhibits abasic site repair by down-regulating APE1 endonuclease activity

Bcl2 not only prolongs cell survival but also suppresses the repair of abasic (AP) sites of DNA lesions. Apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in the repair of AP sites via the base excision repair pathway. Here we found that Bcl2 down-regulates APE1 endonuclease activity in association with inhibition of AP site repair. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus and interaction with APE1, which requires all of the BH domains of Bcl2. Deletion of any of the BH domains from Bcl2 abrogates the ability of Bcl2 to interact with APE1 as well as the inhibitory effects of Bcl2 on APE1 activity and AP site repair. Overexpression of Bcl2 in cells reduces formation of the APE1.XRCC1 complex, and purified Bcl2 protein directly disrupts the APE1.XRCC1 complex with suppression of APE1 endonuclease activity in vitro. Importantly, specific knockdown of endogenous Bcl2 by RNA interference enhances APE1 endonuclease activity with accelerated AP site repair. Thus, Bcl2 inhibition of AP site repair may occur in a novel mechanism by down-regulating APE1 endonuclease activity, which may promote genetic instability and tumorigenesis.     

Characterization of the AP endonucleases from Thermoplasma volcanium and Lactobacillus plantarum: Contributions of two important tryptophan residues to AP site recognition

Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3'-5'exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.     

Effects of backbone contacts 3' to the abasic site on the cleavage and the product binding by human apurinic/apyrimidinic endonuclease (APE1)

The mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) is a multifunctional protein that plays essential roles in DNA repair and gene regulation. We decomposed the APEs into 12 blocks of highly conserved sequence and structure (molegos). This analysis suggested that residues in molegos common to all APEs, but not to the less specific nuclease, DNase I, would dictate enhanced binding to damaged DNA. To test this hypothesis, alanine was substituted for N226 and N229, which form hydrogen bonds to the DNA backbone 3' of the AP sites in crystal structures of the APE1/DNA complex. While the cleavage rate at AP sites of both N226A and N229A mutants increased, their ability to bind to damaged DNA decreased. The ability of a double mutant (N226A/N229A) to bind damaged DNA was further decreased, while the V(max) was almost identical to that of the wild-type APE1. A double mutant at N226 and R177, a residue that binds to the same phosphate as N229, had a significantly decreased activity and substrate binding. As the affinity for product DNA was decreased in all the mutants, the enhanced reaction rate of the single mutants could be due to alleviation of product inhibition of the enzyme. We conclude that hydrogen bonds to phosphate groups 3' to the cleavage site is essential for APE1's binding to the product DNA, which may be necessary for efficient functioning of the base excision repair pathway. The results indicate that the molego analysis can aid in the redesign of proteins with altered binding affinity and activity.     

AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision repair

Base loss is common in cellular DNA, resulting from spontaneous degradation and enzymatic removal of damaged bases. Apurinic/apyrimidinic (AP) endonucleases recognize and cleave abasic (AP) sites during base excision repair (BER). APE1 (REF1, HAP1) is the predominant AP endonuclease in mammalian cells. Here we analyzed the influences of APE1 on the human BER pathway. Specifically, APE1 enhanced the enzymatic activity of both flap endonuclease1 (FEN1) and DNA ligase I. FEN1 was stimulated on all tested substrates, regardless of flap length. Interestingly, we have found that APE1 can also inhibit the activities of both enzymes on substrates with a tetrahydrofuran (THF) residue on the 5'-downstream primer of a nick, simulating a reduced abasic site. However once the THF residue was displaced at least a single nucleotide, stimulation of FEN1 activity by APE1 resumes. Stimulation of DNA ligase I required the traditional nicked substrate. Furthermore, APE1 was able to enhance overall product formation in reconstitution of BER steps involving FEN1 cleavage followed by ligation. Overall, APE1 both stimulated downstream components of BER and prevented a futile cleavage and ligation cycle, indicating a far-reaching role in BER.     

Trypanosoma brucei AP endonuclease 1 has a major role in the repair of abasic sites and protection against DNA-damaging agents

DNA repair mechanisms guarantee the maintenance of genome integrity, which is critical for cell viability and proliferation in all organisms. As part of the cellular defenses to DNA damage, apurinic/apyrimidinic (AP) endonucleases repair the abasic sites produced by spontaneous hydrolysis, oxidative or alkylation base damage and during base excision repair (BER). Trypanosoma brucei, the protozoan pathogen responsible of human sleeping sickness, has a class II AP endonuclease (TBAPE1) with a high degree of homology to human APE1 and bacterial exonuclease III. The purified recombinant enzyme cleaves AP sites and removes 3'-phosphoglycolate groups from 3'-ends. To study its cellular function, we have established TBAPE1-deficient cell lines derived from bloodstream stage trypanosomes, thus confirming that the AP endonuclease is not essential for viability in this cell type under in vitro culture conditions. The role of TBAPE1 in the removal of AP sites is supported by the inverse correlation between the level of AP endonuclease in the cell and the number of endogenously generated abasic sites in its genomic DNA. Furthermore, depletion of TBAPE1 renders cells hypersensitive to AP site and strand break-inducing agents such as methotrexate and phleomycin respectively but not to alkylating agents. Finally, the increased susceptibility that TBAPE1-depleted cells show to nitric oxide suggests an essential role for this DNA repair enzyme in protection against the immune defenses of the mammalian host.     

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease

AP endonuclease (AP endo), a key enzyme in repair of abasic sites in DNA, makes a single nick 5' to the phosphodeoxyribose of an abasic site (AP-site). We recently proposed a novel mechanism, whereby the enzyme uses a key tyrosine (Tyr(171)) to directly attack the scissile phosphate of the AP-site. We showed that loss of the tyrosyl hydroxyl from Tyr(171) resulted in dramatic diminution in enzymatic efficiency. Here we extend the previous work to compare binding/recognition of AP endo to oligomeric DNA with and without an AP-site by wild type enzyme and several tyrosine mutants including Tyr(128), Tyr(171) and Tyr(269). We used single turnover and electrophoretic mobility shift assays. As expected, binding to DNA with an AP-site is more efficient than binding to DNA without one. Unlike catalytic cleavage by AP endo, which requires both hydroxyl and aromatic moieties of Tyr(171), the ability to bind DNA efficiently without an AP-site is independent of an aromatic moiety at position 171. However, the ability to discriminate efficiently between DNA with and without an AP-site requires tyrosine at position 171. Thus, AP endo requires a tyrosine at the active site for the properties that enable it to behave as an efficient, processive endonuclease.