Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed beta-elimination at abasic sites

Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via beta-elimination. An amine serves as the reactive nucleophile to attack C1' on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed beta-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal alpha-amino group, not an epsilon-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed beta-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.     

Substrate specificities and excision kinetics of DNA glycosylases involved in base-excision repair of oxidative DNA damage

Reactive oxygen-derived species such as free radicals are formed in living cells by normal metabolism and exogenous sources, and cause a variety of types of DNA damage such as base and sugar damage, strand breaks and DNA-protein cross-links. Living organisms possess repair systems that repair DNA damage. Oxidative DNA damage caused by free radicals and other oxidizing agents is mainly repaired by base-excision repair (BER), which involves DNA glycosylases in the first step of the repair process. These enzymes remove modified bases from DNA by hydrolyzing the glycosidic bond between the modified base and the sugar moiety, generating an apurinic/apyrimidinic (AP) site. Some also possess AP lyase activity that subsequently cleaves DNA at AP sites. Many DNA glycosylases have been discovered and isolated, and their reaction mechanisms and substrate specificities have been elucidated. Most of the known products of oxidative damage to DNA are substrates of DNA glycosylases with broad or narrow substrate specificities. Some possess cross-activity and remove both pyrimidine- and purine-derived lesions. Overlapping activities between enzymes also exist. Studies of substrate specificities have been performed using either oligodeoxynucleotides with a single modified base embedded at a specific position or damaged DNA substrates containing a multiplicity of pyrimidine- and purine-derived lesions. This paper reviews the substrate specificities and excision kinetics of DNA glycosylases that have been investigated with the use of gas chromatography/mass spectrometry and DNA substrates with multiple lesions.     

Affinity modification in a proteomic study of DNA repair ensembles

The review concerns the use of the affinity modification method as an integral part of the modern proteomic analysis to search for and identification of proteins belonging to protein ensembles of DNA repair. Affinity modification is based on the preliminary formation of specific non-covalent complex between the target biopolymer and a reagent (chemically reactive analog of biopolymer or low molecular weight ligand) followed by formation of covalent bond between the reagent and the site of the target, to which the reagent is bound, that ensures the method specificity. This method is most widely and effectively used in the study of structural and functional aspects of protein-nucleic acids interactions. Upon construction of DNA probes, in addition to chemically reactive groups and structural elements involved in specific recognition of DNA by proteins, additional groups that facilitate the subsequent affinity isolation of DNA-protein cross-links, can be introduced into the reagent. The review covers recent examples affinity DNA-reactive probe in combination with mass spectrometric and immunological methods to search for and identification in cell extracts, proteins interacting with apurinic/apyrimidinic sites and the proteins recognizing the cross-links in DNA induced by cisplatin.     

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.     

Base-excision repair of oxidative DNA damage by DNA glycosylases

Oxidative damage to DNA caused by free radicals and other oxidants generate base and sugar damage, strand breaks, clustered sites, tandem lesions and DNA-protein cross-links. Oxidative DNA damage is mainly repaired by base-excision repair in living cells with the involvement of DNA glycosylases in the first step and other enzymes in subsequent steps. DNA glycosylases remove modified bases from DNA, generating an apurinic/apyrimidinic (AP) site. Some of these enzymes that remove oxidatively modified DNA bases also possess AP-lyase activity to cleave DNA at AP sites. DNA glycosylases possess varying substrate specificities, and some of them exhibit cross-activity for removal of both pyrimidine- and purine-derived lesions. Most studies on substrate specificities and excision kinetics of DNA glycosylases were performed using oligonucleotides with a single modified base incorporated at a specific position. Other studies used high-molecular weight DNA containing multiple pyrimidine- and purine-derived lesions. In this case, substrate specificities and excision kinetics were found to be different from those observed with oligonucleotides. This paper reviews substrate specificities and excision kinetics of DNA glycosylases for removal of pyrimidine- and purine-derived lesions in high-molecular weight DNA.     

Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair

Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.     

Interaction of nucleotide excision repair proteins with DNA containing bulky lesion and apurinic/apyrimidinic site

The interaction of nucleotide excision repair (NER) proteins (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes containing the bulky lesion-mimicking fluorescein-substituted derivative of dUMP (5-{3-[6-(carboxyamidofluo-resceinyl)amidocapromoyl]allyl}-2′-deoxyuridine-5′-monophosphate) in a cluster with a lesion of another type (apurinic/apyrimidinic (AP) site) has been studied. It is shown that XPC-HR23b is modified to a greater extent by the DNA duplex containing an AP site opposite nucleotide adjacent to the fluorescein residue than by DNA containing an AP site shifted to the 3′-or 5′-end of the DNA strand. The efficiency of XPA modification by DNA duplexes containing both AP site and fluorescein residue is higher than that by DNA lacking the bulky lesion; the modification pattern in this case depends on the AP site position. In accordance with its major function, RPA interacts more efficiently with single-stranded DNA than with DNA duplexes, including those bearing bulky lesions. The observed interaction between the proteins involved in nucleotide excision repair and DNA structures containing a bulky lesion processed by NER and the AP site repaired via base excision repair may be significant for both these repair pathways in cells and requires the specific sequence of repair of clustered DNA lesions.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

Interaction of nucleotide excision repair protein XPC—RAD23B with DNA containing benzo[a]pyrene-derived adduct and apurinic/apyrimidinic site within a cluster

The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms — NER and BER, respectively. Interaction of NER protein XPC—RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N²-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC—RAD23B crosslinks to DNA containing (+)-trans-BPDE-N²-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N²-dG isomers depends on the AP site position in the opposite strand. The influence of XPC—RAD23B on hydrolysis of an AP site clustered with BPDE-N²-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC—RAD23B was shown to stimulate the endonuclease and inhibit the 3′–5′ exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage.     

Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV

Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N(2)-N(2)-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5' to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3' to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N(2)-N(2)-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N(2)-N(2)-guanine ICLs.     

Preferential binding of human full-length XPA and the minimal DNA binding domain (XPA-MF122) with the mitomycin C-DNA interstrand cross-link

Nucleotide excision repair (NER) is an important cellular mechanism that removes radiation-induced and chemically induced damage from DNA. The XPA protein is involved in the damage recognition step of NER and appears to function by binding damaged DNA and recruiting other proteins to the site. It may also play a role in subsequent steps of NER through interaction with other repair proteins. Interstrand cross-links are of particular interest, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery. Using 14 and 25 bp duplex oligonucleotides containing a defined, well-characterized single mitomycin C (MMC)-DNA interstrand cross-link, we have shown through gel shift analysis that both XPA and a minimal DNA binding domain of XPA (XPA-MF122) preferentially bind to MMC-cross-linked DNA with a greater specificity and a higher affinity (>2-fold) than to the same undamaged DNA sequence. This preferential binding to MMC-cross-linked DNA occurs in the absence of other proteins from the NER complex. Differences in binding affinity and specificity were observed among the different protein-DNA combinations that were both protein and DNA specific. Defining XPA-MMC-DNA interactions may aid in elucidating the mechanism by which DNA cross-links and other forms of DNA damage are recognized and repaired by the NER machinery in eukaryotic cells.     

Mutational analysis of the human nucleotide excision repair gene ERCC1.

The human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic recombination pathway, probably required for repair of DNA cross-links. Mutational analysis revealed that the poorly conserved N-terminal 91 amino acids of ERCC1 are dispensable for both repair functions, in contrast to a deletion of only four residues from the C-terminus. A database search revealed a strongly conserved motif in this C-terminus sharing sequence homology with many DNA break processing proteins, indicating that this part is primarily required for the presumed structure-specific endonuclease activity of ERCC1. Most missense mutations in the central region give rise to an unstable protein (complex). Accordingly, we found that free ERCC1 is very rapidly degraded, suggesting that protein-protein interactions provide stability. Survival experiments show that the removal of cross-links requires less ERCC1 than UV repair. This suggests that the ERCC1-dependent step in cross-link repair occurs outside the context of NER and provides an explanation for the phenotype of the human repair syndrome xeroderma pigmentosum group F.     

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.     

Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.     

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.     

Processing of a psoralen DNA interstrand cross-link by XPF-ERCC1 complex in vitro

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5' to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3'-side of an ICL. The ICL-specific 3'-incision, along with the 5'-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.     

Closely opposed apurinic/apyrimidinic sites are converted to double strand breaks in Escherichia coli even in the absence of exonuclease III, endonuclease IV, nucleotide excision repair and AP lyase cleavage

Multiply damaged sites (MDSs) consist of two or more damages within 20 base pairs (bps) and are introduced into DNA by ionizing radiation. Using a plasmid assay, we previously demonstrated that repair in Escherichia coli generated a double strand break (DSB) from two closely opposed uracils when uracil DNA glycosylase initiated repair. To identify the enzymes that converted the resulting apurinic/apyrimidinic (AP) sites to DSBs, repair was examined in bacteria deficient in AP site cleavage. Since exonuclease III (xth) and endonuclease IV (nfo) mutant bacteria were able to introduce DSBs at the MDSs, we generated unique bacterial mutants deficient in UvrA, Xth and Nfo. However, the additional disruption of nucleotide excision repair (NER) did not prevent DSB formation. xth- nfo- nfi- bacteria also converted the MDSs to DSBs, ruling out endonuclease V as the candidate AP endonuclease. By using MDSs containing tetrahydrofuran (an AP site analog), it was determined that even in the absence of Xth, Nfo, NER and AP lyase cleavage, DSBs were formed from closely opposed AP sites. This finding implies that there is an unknown enzyme/repair pathway for MDSs, and multiple underlying repair systems in cells that can process closely opposed DNA damage into lethal lesions following exposure to ionizing radiation.     

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.     

Kinetic mechanism of the interaction of Saccharomyces cerevisiae AP-endonuclease 1 with DNA substrates

The apurinic/apyrimidinic endonuclease from Saccharomyces cerevisiae Apn1 is one of the key enzymes involved in base excision repair of DNA lesions. A major function of the enzyme is to cleave the upstream phosphodiester bond of an apurinic/apyrimidinic site (AP-site), leading to the formation of a single-strand break with 3′-hydroxyl (OH) and 5′-deoxyribose phosphate (dRP) termini. In this study, the pre-steady-state kinetics and conformational dynamics of DNA substrates during their interaction with Apn1 were investigated. A stopped-flow method with detection of the fluorescence intensity of 2-aminopurine and pyrrolocytosine located adjacent or opposite to the damage was used. It was found that upon interaction with Apn1, both DNA strands undergo a number of rapid changes. The location of fluorescent analogs of heterocyclic bases in DNA does not influence the catalytic step of the reaction. Comparison of data obtained for yeast Apn1 and reported data (Kanazhevskaya, L. Yu., Koval, V. V., Vorobjev, Yu. N., and Fedorova, O. S. (2012) Biochemistry, 51, 1306–1321) for human Ape1 revealed some differences in their interaction with DNA substrates.