Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.     

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.     

The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, Chlorella virus-pdg

The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Site-directed mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed.     

Characterization of a wide range base-damage-endonuclease activity of mammalian rpS3

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.     

AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe

Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

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.     

DNA repair in cultured mouse cells of increasing population doubling level

Cultures of mouse cells of various population doubling levels (PDL) were examined for DNA-repair capabilities as estimated by (i) the excision of pyrimidine dimers; (ii) unscheduled DNA synthesis (UDS) in response to UV-irradiation or N-methyl-N'-nitrosoguanidine (MNNG) treatment; (iii) the levels of two DNA-repair enzyme activities, uracil DNA glycosylase and AP endonuclease. The responses to ultraviolet light and MNNG decreased rapidly within the first two PDL and more slowly thereafter until essentially no repair was detected by PDL 12. A continuous cell line which emerged from the cultured cells after a crises period had some restoration of repair capability. The amount of uracil DNA glycosylase activity decreased by approximately 40% before the crises period then decreased by 90% in the continuous cell line. In contrast, the amount of AP endonuclease activity present in the precrises cells showed no significant change until PDL 12, then increased 6-7-fold in the continuous cell line.     

DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway

Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway. The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated. We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities. This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides. Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway. This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway. The apparent absence of a short-patch BER pathway in P. falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells.     

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

Repair of depurinated DNA with enzymes from rat liver chromatin.

DNA from T7 phage containing AP (apurinic/apyrimidinic) sites was repaired by the successive actions of three chromatin enzymes [AP endodeoxyribonuclease, DNAase IV (5'----3'-exodeoxyribonuclease) and DNA polymerase-beta] prepared from rat liver and T4-phage DNA ligase. Since DNA ligase is also found in rat liver chromatin, all the activities used for the successful repair in vitro are thus present in the chromatin of a eukaryotic cell. Our results show, in particular, that the chromatin DNAase IV is capable of excising the AP site from the DNA strand nicked by the chromatin AP endodeoxyribonuclease. We did not try to combine all the enzymes, since competition between some of them might have prevented the repair; we have, for instance, shown that DNA ligase can seal the incision 5' to the AP site made by the AP endodeoxyribonuclease. Changes in chromatin structure during repair might perhaps prevent this competition when nuclear DNA is repaired in the living cell.     

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.     

The phosphonomethyl analogue of 3-phosphoglycerate is a potent competitive inhibitor of phosphoglycerate mutases.

Deoxyribonuclease IV, a 5'-3' exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5' to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3' to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.     

Repair of deaminated bases in DNA

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine. When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed. The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others. The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases. In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap. The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase. DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells. Many of these glycosylases have been cloned and well characterized. In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine. In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells. In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V. Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion. It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base. The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase. Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.     

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) β and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3′-terminal sugar phosphate by the 3′-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol β were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol β strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol β and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.     

Action of Escherichia coli and human 5'----3' exonuclease functions at incised apurinic/apyrimidinic sites in DNA.

The 5'----3' exonuclease activity of E. coli DNA polymerase I and a related enzyme activity in mammalian cell nuclei, DNase IV, are unable to catalyse the excision of free deoxyribose-phosphate from apurinic/apyrimidinic (AP) sites incised by an AP endonuclease. Instead, the sugar phosphate residue is slowly released as part of a short oligonucleotide. These products have been characterised as dimers and trimers by comparison of their retention time on reverse-phase HPLC with reference compounds prepared by acid depurination of a dinucleotide, trinucleotide and tetranucleotide containing a 5'-terminal dAMP residue. The similar mode of action of these enzymes at 5'-incised AP sites provides an explanation for the minority of repair patches larger than one nucleotide observed when AP sites are repaired by E. coli and mammalian cell extracts in vitro and strengthens the functional analogy between the two activities.     

Activities involved in base excision repair of bacteriophage T4 and lambda DNA in vivo

Summary The in vivo excision repair functions of Escherichia coli exonuclease III and 3-methyladenine DNA glycosylase I, and bacteriophage T4 pyrimidine dimer-DNA glycosylase were investigated. Following exposure of bacteriophage T4 or lambda to methyl methanesulfonate or ultraviolet irradiation, survival was determined by plating on E. coli have various genetic backgrounds. Although exonuclease III was shown to participate in base excision repair initiated by 3-methyladenine DNA glcosylase I, it had no detectable role in base excision repair initiated by the T4 pyrimidine dimer-DNA glycosylase. Despite its 3 apurinic/apyrimidinic endonuclease activity in vitro, T4 pyrimidine dimer-DNA glycosylase, even in large quantities, did not complement mutants defective in exonuclease III in the repair of apurinic sites generated by 3-methyladenine DNA glycosylase I in vivo.     

Replication protein A stimulates proliferating cell nuclear antigen-dependent repair of abasic sites in DNA by human cell extracts.

Base excision repair (BER) pathway is the major cellular process for removal of endogenous base lesions and apurinic/apyrimidinic (AP) sites in DNA. There are two base excision repair subpathways in mammalian cells, characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" (one nucleotide incorporated) and the "long-patch" (several nucleotides incorporated) BER pathways. Proliferating cell nuclear antigen (PCNA) is known to be an essential factor in long-patch base excision repair. We have studied the role of replication protein A (RPA) in PCNA-dependent, long-patch BER of AP sites in human cell extracts. PCNA and RPA were separated from the other BER proteins by fractionation of human whole-cell extract on a phosphocellulose column. The protein fraction PC-FII (phosphocellulose fraction II), which does not contain RPA and PCNA but otherwise contains all core BER proteins required for PCNA-dependent BER (AP endonuclease, DNA polymerases delta, beta and DNA ligase, and FEN1 endonuclease), had reduced ability to repair plasmid DNA containing AP sites. Purified PCNA or RPA, when added separately, could only partially restore the PC-FII repair activity of AP sites. However, additions of both proteins together greatly stimulated AP site repair by PC-FII. These results demonstrate a role for RPA in PCNA-dependent BER of AP sites.     

Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

The UV endonucleases [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. We have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV- (4 kJ/m2, 254 nm) treated, [3H]thymine-labeled poly(dA).poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical- (5 kJ/m2, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. Our data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. Our results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.