DNA polymerase beta and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair

DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) are key players in pol beta-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol beta strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol beta DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5'-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol beta-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol beta substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol beta DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol beta. This study demonstrates that pol beta cooperates with FEN1 to remove DNA damage via a "Hit and Run" mechanism, involving alternating short gap production by FEN1 and gap filling by pol beta, rather than through coordinated formation and removal of a strand-displaced flap.     

Interaction of human AP endonuclease 1 with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-patch base excision repair

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.     

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.     

Long patch base excision repair with purified human proteins. DNA ligase I as patch size mediator for DNA polymerases delta and epsilon

Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases delta or epsilon, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3' to the lesion. However, almost 70% of the repair synthesis was confined to 2-4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.     

Role of DNA polymerase beta in the excision step of long patch mammalian base excision repair.

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta (pol beta) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta. We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta null cells and is rescued by addition of purified pol beta. Also, pol beta-neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.     

Nucleosome disruption by DNA ligase III-XRCC1 promotes efficient base excision repair

Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase β (Pol β), which catalyze the first three steps in BER, are able to process their substrates in both 601- and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase IIIα-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase IIIα-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol β on nucleosome substrates. Collectively, these findings provide insights into rate-limiting steps that govern BER in chromatin and reveal a unique role for ligase IIIα-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes.     

AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair

Base excision repair (BER) of an oxidized base within a trinucleotide repeat (TNR) tract can lead to TNR expansions that are associated with over 40 human neurodegenerative diseases. This occurs as a result of DNA secondary structures such as hairpins formed during repair. We have previously shown that BER in a TNR hairpin loop can lead to removal of the hairpin, attenuating or preventing TNR expansions. Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3′-5′ exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop. Coordinating with flap endonuclease 1, the APE1 3′-5′ exonuclease activity cleaves the annealed upstream 3′-flap of a double-flap intermediate resulting from 5′-incision of an abasic site in the hairpin loop. Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.     

ATP-dependent selection between single nucleotide and long patch base excision repair

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.     

The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta in long patch base excision repair

Growing evidence suggests that the Rad9-Rad1-Hus1 complex (the 9-1-1 complex), besides its functions in DNA damage sensing and signaling pathways, plays also a direct role in various DNA repair processes. Recent studies have demonstrated that the 9-1-1 complex physically and functionally interacts with several components of the base excision repair (BER) machinery namely DNA polymerase β (Pol β), flap endonuclease 1 (Fen 1), DNA ligase I (Lig I) and the MutY homologue of Schizosaccharomyces pombe. In this work, we found for the first time that the 9-1-1 complex interacts in vitro and in vivo with the apurinic/apyrimidinic endonuclease 1 (APE 1), an early component of BER, and can stimulate its AP-endonuclease activity. Moreover, we show that the 9-1-1 complex possesses a stimulatory effect on long patch base excision repair (LP-BER) reconstituted in vitro. The enhancement of LP-BER activity is due to the specific stimulation of the two early components of the repair machinery, namely APE 1 and Pol β, suggesting a hierarchy of interactions between the 9-1-1 complex and the BER proteins acting in the repairosome. Overall, our results indicate that the 9-1-1 complex is directly involved in LP-BER, thus providing a possible link between DNA damage checkpoints and BER.     

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.     

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ss (ss-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3'- to 5'-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5'-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.     

Inhibition of short patch and long patch base excision repair by an oxidized abasic site

5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents, including several antitumor antibiotics. DOB efficiently and irreversibly inhibits DNA polymerase β, an essential base excision repair enzyme in mammalian cells. The generality of this mode of inhibition by DOB is supported by the inactivation of DNA polymerase λ, which may serve as a possible backup for DNA polymerase β during abasic site repair. Protein digests suggest that Lys72 and Lys84, which are present in the lyase active site of DNA polymerase β, are modified by DOB. Monoaldehyde analogues of DOB substantiate the importance of the 1,4-dicarbonyl component of DOB for efficient inactivation of Pol β and the contribution of a freely diffusible electrophile liberated from the inhibitor by the enzyme. Inhibition of DNA polymerase β's lyase function is accompanied by inactivation of its DNA polymerase activity as well, which prevents long patch base excision repair of DOB. Overall, DOB is highly refractory to short patch and long patch base excision repair. Its recalcitrance to succumb to repair suggests that DOB is a significant source of the cytotoxicity of DNA damaging agents that produce it.     

Flap endonuclease 1 efficiently cleaves base excision repair and DNA replication intermediates assembled into nucleosomes

Flap Endonuclease 1 (FEN1) plays important roles both in DNA replication and in base excision repair (BER). However, in both processes FEN1 substrates are likely to be assembled into chromatin. In order to examine how FEN1 is able to work within chromatin, we prepared model nucleosome substrates containing FEN1-cleavable DNA flaps. We find that human FEN1 binds and cleaves such substrates with efficiencies similar to that displayed with naked DNA. Moreover, we demonstrate that both FEN1 and human DNA ligase I can operate successively on DNA within the same nucleosome. These results suggest that some BER steps may not require nucleosome remodeling in vivo and that FEN 1 activity during Okazaki fragment processing can occur on nucleosomal substrates.     

DNA polymerase beta -mediated long patch base excision repair. Poly(ADP-ribose)polymerase-1 stimulates strand displacement DNA synthesis.

Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase.

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

DNA polymerase beta promotes recruitment of DNA ligase III alpha-XRCC1 to sites of base excision repair

Base excision repair is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). Base excision repair requires the coordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. Using a protein-DNA cross-linking assay during repair in human whole cell extracts, we monitored proteins involved in the initial steps of repair of a substrate containing a site-specific abasic site to address the molecular events following incision of the abasic site by AP endonuclease. We find that after dissociation of AP endonuclease from the incised abasic site, both DNA polymerase beta (Pol beta) and the DNA ligase IIIalpha-XRCC1 heterodimer efficiently bind/cross-link to the substrate DNA. We also find that the cross-linking efficacy of the DNA ligase IIIalpha-XRCC1 heterodimer was decreased about 2-fold in the Pol beta-deficient cell extract but was rescued by addition of purified wild type but not a mutant Pol beta protein that does not interact with the DNA ligase IIIalpha-XRCC1 heterodimer. We further demonstrate that Pol beta and the DNA ligase IIIalpha-XRCC1 heterodimer are present at equimolar concentrations in whole cell extracts and that Pol beta has a 7-fold higher affinity to the incised abasic site containing substrate than DNA ligase IIIalpha. Using gel filtration of whole cell extracts prepared at physiological salt conditions (0.15 M NaCl), we find no evidence for a stable preexisting complex of DNA Pol beta with the DNA ligase IIIalpha-XRCC1 heterodimer. Taken together, these data suggest that following incision by AP endonuclease, DNA Pol beta recognizes and binds to the incised abasic site and promotes recruitment of the DNA ligase IIIalpha-XRCC1 heterodimer through its interaction with XRCC1.     

DNA polymerase beta-catalyzed-PCNA independent long patch base excision repair synthesis: a mechanism for repair of oxidatively damaged DNA ends in post-mitotic brain

Oxidative DNA damage incidental to normal respiratory metabolism poses a particular threat to genomes of highly metabolic-long lived cells. We show that post-mitotic brain has capacity to repair oxidatively damaged DNA ends, which are targets of the long patch (LP) base excision repair (BER) subpathway. LP-BER relies, in part, on proteins associated with DNA replication, including proliferating cell nuclear antigen and is inherent to proliferating cells. Nonetheless, repair products are generated with brain extracts, albeit at slow rates, in the case of 5'-DNA ends modeled with tetrahydrofuran (THF). THF at this position is refractory to DNA polymerase beta 5'-deoxyribose 5-phosphate lyase activity and drives repair into the LP-BER subpathway. Comparison of repair of 5'-THF-blocked termini in the post-mitotic rat brain and proliferative intestinal mucosa, revealed that in mucosa, resolution of damaged 5'-termini is accompanied by formation of larger repair products. In contrast, adducts targeted by the single nucleotide BER are proficiently repaired with both extracts. Our findings reveal mechanistic differences in BER processes selective for the brain versus proliferative tissues. The differences highlight the physiological relevance of the recently proposed 'Hit and Run' mechanism of alternating cleavage/synthesis steps, in the proliferating cell nuclear antigen-independent LP-BER process.