Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation.

The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.     

Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA.

Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.     

Characterization of Excision Repair in Neurospora crassa

The excision of pyrimidine dimers from the deoxyribonucleic acid (DNA) of Neurospora crassa was examined. Postirradiation incubation in the presence of several chemicals known to inhibit various repair systems indicated that caffeine reduced the rate of excision twofold, but did not inhibit excision completely as did proflavine and quinacrine. Examination of the time course of excision showed that repair occurs at a relatively rapid rate: approximately 60 dimers excised per min after 500 ergs/mm(2). Further evidence for rapid excision was obtained by sedimentation analysis of DNA; the maximal number of breaks introduced during repair was three, suggesting that breaks are repaired almost as fast as they are made and that only a few dimers are repaired at a time. Repair synthesis was measured by prelabeling the DNA with (15)N and D(2)O, and then subjecting the DNA to equilibrium density gradient centrifugation after postirradiation incubation with (32)P. Accumulation of single-strand breaks with increasing dose of ultraviolet radiation suggested that the limiting step was subsequent to the incision and excision steps of repair. Equilibrium CsCl centrifugation demonstrated that the limiting step in excision was repair synthesis.     

Evidence in Escherichia coli that N3-methyladenine lesions induced by a minor groove binding methyl sulfonate ester can be processed by both base and nucleotide excision repair

It has been previously reported that a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (MeOSO(2)-lex) selectively affords N3-methyladenine lesions. To study the interaction of the neutral lex dipeptide with calf thymus DNA, we have prepared stable, nonmethylating sulfone analogues of MeOSO(2)-lex that are neutral and cationic. Thermodynamic studies show that both the neutral and monocationic sulfone compounds bind to DNA with K(b)'s of 10(5) in primarily entropy-driven reactions. To determine how the cytotoxic N3-methyladenine adduct generated from MeOSO(2)-lex is repaired in E. coli, MeOSO(2)-lex was tested for toxicity in wild-type E. coli and in mutant strains defective in base excision repair (tag and/or alkA glycosylases or apn endonuclease), nucleotide excision repair (uvrA), and both base and nucleotide excision repair (tag/alkA/uvrA). The results clearly demonstrate the cellular toxicity of the N3-methyladenine lesion, and the protective role of base excision glycosylase proteins. A novel finding is that in the absence of functional base excision glycosylases, nucleotide excision repair can also protect cells from this cytotoxic minor groove lesion. Interaction between base and nucleotide excision repair systems is also seen in the protection of cells treated with cis-diamminedichloroplatinum(II) but not with anti-(+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.     

Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutières syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays, we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in cultured human cells. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells.     

A sensitive flow cytometry-based nucleotide excision repair assay unexpectedly reveals that mitogen-activated protein kinase signaling does not regulate the removal of UV-induced DNA damage in human cells

In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.     

A review of recent experiments on step-to-step "hand-off" of the DNA intermediates in mammalian base excision repair pathways

The current "working model" for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the base excision repair intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In this review, we explore the question of whether there is an actual step-to-step "hand-off" of the DNA intermediates during base excision repair in vitro. The results show that when base excision repair enzymes are pre-bound to the initial single-nucleotide base excision repair intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase beta and then to DNA ligase. In the long patch base excision repair sub-pathway, where the 5'-end of the incised strand is blocked, the intermediate after polymerase beta gap filling is not channeled from polymerase beta to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.     

A review of recent experiments on step-to-step “hand-off” of the DNA intermediates in mammalian base excision repair pathways

The current “working model” for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the base excision repair intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In this review, we explore the question of whether there is an actual step-to-step “hand-off” of the DNA intermediates during base excision repair in vitro. The results show that when base excision repair enzymes are pre-bound to the initial single-nucleotide base excision repair intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch base excision repair sub-pathway, where the 5′-end of the incised strand is blocked, the intermediate after polymerase β gap filling is not channeled from polymerase β to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase beta, and ligases, require Mg(2+). Here we have demonstrated that Mg(2+) can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg(2+) at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg(2+) affected K(m), but not V(max), and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg(2+) stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg(2+) inhibits MPG activity by abrogating substrate binding. Because Mg(2+) is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.     

Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain. We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2). In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place. This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant. Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.     

Nitric oxide inhibits DNA-adduct excision in nucleotide excision repair

Sustained induction of nitric oxide (NO) in chronic inflammation may be mutagenic, through DNA damage induction and/or DNA repair inhibition. Although there is good evidence that NO can cause DNA damage, how NO is involved in DNA repair remains elusive. By using DNA synthesis inhibitors to accumulate DNA strand breaks in comet assay, we show that NO and peroxynitrite inhibit DNA-adduct excision in human fibroblasts damaged by UVC, 4-nitroquinoline 1-oxide, benzo[a]pyrene dihydrodiol epoxide, cisplatin, or mitomycin C, but not with methyl methane sulfonate. Treating cells with arsenite increased NO production and also inhibited the DNA-adduct excision induced by UVC, 4-nitroquinoline 1-oxide, benzo[a]pyrene dihydrodiol epoxide, cisplatin, and mitomycin C, but not by methyl methane sulfonate, H(2)O(2), sodium nitrosoprusside, or 3-morpholinosydnonimine. Arsenite inhibition of DNA-adduct excision was decreased by NO synthase inhibitors and NO scavengers. The nuclear extract prepared from fibroblasts pretreated with sodium nitrosoprusside, dipropylenetriamine NONOate, 3-morpholinosydnonimine, or arsenite also showed decreased activity in excising the DNA adducts induced by UVC and cisplatin but not by methyl methane sulfonate or H(2)O(2) plus Fe. These results are consistent with the notion that NO, peroxynitrite, and arsenite inhibit the DNA-adduct excision in nucleotide excision repair but not that in base excision repair.     

Effects of hyperthermia on radiation-induced chromosome breakage and loss in excision repair deficient Drosophila melanogaster

Hyperthermia increased radiosensitivity with respect to gamma-ray induced chromosome loss and breakage in all stages of spermatogenesis in the wild type Oregon R strain of Drosophila melanogaster, whereas hyperthermia increased radiosensitivity to a lesser extent in cn mus (2) 201D1, an excision repair mutant with 0 per cent excision capacity and in mus (3) 308D1, a strain with 24 per cent excision capacity. The differences in hyperthermia-induced radiation sensitivity between the excision repair mutants and the wild strain may be due to the hyperthermia affecting the excision repair mechanism, suggesting that one of the possible mechanisms involved in hyperthermia-increased radiosensitivity is an effect on excision repair.     

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.