Excision Of Pyrimidine Dimers Research Articles

LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage.

Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4)and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery.

HDAC3 Is Required for XPC Recruitment and Nucleotide Excision Repair of DNA Damage Induced by UV Irradiation.

Recent studies have demonstrated that lysine acetylation of histones is crucial for nucleotide excision repair (NER) by relaxing the chromatin structure, which facilitates the recruitment of repair factors. However, few studies have focused on the contribution of histone deacetylases (HDAC) to NER. Here, we found that histone H3 Lys14 (H3K14) was deacetylated by HDAC3 after UV irradiation. Depletion of HDAC3 caused defects in cyclobutene pyrimidine dimer excision and sensitized cells to UV irradiation. HDAC3-depleted cells had impaired unscheduled DNA synthesis, but not recovery of RNA synthesis, which indicates that HDAC3 was required for global genome NER. Moreover, xeroderma pigmentosum, complementation group C (XPC) accumulation at the local UV-irradiated area was attenuated in HDAC3-depleted cells. In addition to the delay of XPC accumulation at DNA damage sites, XPC ubiquitylation was inhibited in HDAC3-depleted cells. These results suggest that the deacetylation of histone H3K14 by HDAC3 after UV irradiation contributes to XPC recruitment to DNA lesions to promote global genome NER. IMPLICATIONS: Involvement of histone deacetylation for XPC accumulation after UV irradiation indicates conversion of chromatin structure is essential for nucleotide excision repair in human cancer cells.

Identification of low-molecular-weight vitellogenin 1 (Vg1)-like proteins as nucleotide excision repair (NER) factors in developing zebrafish (Danio rerio) using a transcription-based DNA repair assay.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions such as UV-induced pyrimidine dimers and cisplatin-induced strand crosslinking. Our earlier studies have identified low-molecular-weight proteins homologous to the 150-kDa vitellogenin 1 (Vg1) as UV-damaged DNA-binding factors expressed in developing zebrafish (Danio rerio). This present study explored if Vg1-like proteins also participated in NER in zebrafish. Immunoblot analysis of affinity-captured 12h post-fertilization (hpf) zebrafish extract proteins showed a transient binding of a 30-kDa Vg1-like polypeptide to UV-damaged DNA. A transcription-based in vitro repair assay revealed a significant up-regulation of UVC or cisplatin-suppressed transcriptional activity of a marker cDNA driven by a SP6 RNA polymerase-regulated promotor after incubating the damaged plasmid with the extracts of 12 hpf embryos or 96 hpf larvae. The up-regulation of UV or cisplatin-suppressed transcription was abolished in the presence of a monoclonal anti-zebrafish Vg1 antibody. The differential sensitivity of UV-induced repair in 12 and 96 hpf zebrafish extracts to exogenous ATP suggested a development-dependent expression of Vg1-like NER factors. A T4 endonuclease V digestion assay showed no inhibition of the anti-Vg1 antibody on the excision of UV-induced cyclobutane pyrimidine dimers. Our results identified the participation of Vg1-like factors in NER in developing zebrafish, and these factors may function at post-incison steps of NER.

Inactivation of nucleotide excision repair in Chinese hamster ovary cells by exposure to an alkylating agent.

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μM) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.

XPC lymphoblastoid cells defective in the hMutSα DNA mismatch repair complex exhibit normal sensitivity to UVC radiation and normal transcription-coupled excision repair of DNA cyclobutane pyrimidine dimers

Nucleotide excision (NER) is generally considered to comprise two partially distinct subpathways. Global genomic repair (GGR) removes damage from the genome overall and transcription-coupled repair (TCR) selectively excises damage from transcribed DNA. Cells from individuals belonging to xeroderma pigmentosum (XP) complementation group C are defective in GGR but retain a functional TCR pathway. DNA mismatch repair (MMR) corrects replication errors but can also process DNA damage. It has been suggested that the essential hMutSα and hMutLα MMR protein complexes are also required for effective excision of UV-induced cyclobutane pyrimidine dimers (CPD) by TCR. We have combined an MMR and an XPC defect in a human lymphoblastoid cell line. The MMR-defective XPC cells were defective in the hMutSα mismatch recognition complex that comprises hMSH2 and hMSH6. They were not detectably more sensitive to killing by UV than their MMR proficient counterparts and were able to excise CPDs from an actively transcribed DNA strand. We conclude efficient TCR does not depend on a functional hMutSα complex.

REX1, a Novel Gene Required for DNA Repair

Nucleotide excision repair is a major pathway for repairing UV light-induced DNA damage in most organisms. Using insertional mutagenesis, we isolated a UV-sensitive mutant of Chlamydomonas reinhardtii that is blocked in the excision of cyclobutane pyrimidine dimers. The mutant is also sensitive to the alkylating agent, methyl methanesulphonate. We have cloned REX1, a novel gene that rescues the mutant. The gene is unusual in a eukaryotic organism in that it is predicted to encode two different proteins, a small protein (8.9 kDa) and a larger protein (31.8 kDa). Neither protein is homologous to known DNA repair proteins. Partial complementation is achieved with subclones of the gene encoding only the 8.9-kDa protein. The 8.9-kDa protein has homologues in many organisms including Saccharomyces cerevisiae, Arabidopsis, and humans. The 31.8-kDa protein appears to be less conserved. These findings may be of general importance for DNA repair in other organisms.

Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision.

Arsenite is a human carcinogen reported to inhibit DNA repair. The binding of arsenite to functional thiol groups of DNA repair enzymes has in the past been suggested as a possible mechanism for the effect of arsenite on DNA repair. However, recent studies indicate that reactive oxygen species and nitric oxide are involved in arsenite toxicity. This research aims to elucidate the role of these possible mechanisms in the inhibition of UV-induced DNA repair by arsenite. As arsenite inhibits UV-DNA repair in Chinese hamster ovary cells, and this is a commonly used cell line for UV repair experiments, we used these cells to examine the effect of arsenite on the expression of UV-irradiated reporter genes. The T4 UV endonuclease V-incorporated comet assay was used to examine specifically the effect of arsenite on pyrimidine dimer excision. We showed that inhibition of UV-DNA repair by arsenite was suppressed by nitric oxide synthase inhibitors. Arsenite increased nitric oxide production and nitric oxide generators inhibited UV-DNA repair. The involvement of nitric oxide in the inhibition of pyrimidine dimer excision by arsenite was also confirmed in human fibroblasts. Investigation into the effect of oxidant modulators did not give a clear indication that reactive oxygen species are involved in arsenite inhibition of UV-DNA repair. Phenylarsine oxide, a strong thiol-reacting agent, did not inhibit pyrimidine dimer excision and also did not increase nitric oxide production. Our results show conclusively that nitric oxide is involved in the inhibition of pyrimidine dimer excision by arsenite. Reactive oxygen species and the binding of arsenite to functional thiol groups of DNA repair enzymes do not appear to be involved.

Damaged DNA-binding Protein DDB Stimulates the Excision of Cyclobutane Pyrimidine Dimers in Vitro in Concert with XPA and Replication Protein A

Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6-4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA + replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA + RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA.RPA, and thus stimulates the excision reaction of CPDs.

In vivo induction of pyrimidine dimers in human skin by UVA radiation: initiation of cell damage and/or intercellular communication?

Pyrimidine dimers participate as important factors in ultraviolet-induced lethality, mutagenicity and tumorgenicity. Substantial efforts have been made in recent years to understand the induction of pyrimidine photodimers and their repair in human skin cells exposed to low physiological fluences of UV-light. Dimers are known to be efficiently induced after UVC and UVB irradiation, but these photoproducts are also highly induced in DNA isolated from human skin irradiated with UVA. By using a sensitive immunohistochemistry dimer detection assay, we confirm that in vivo UVA radiation induces substantial amounts of these DNA changes in the epidermis; in addition, this technique detects them far into the reticular dermis. A considerable number of these photodimers were also seen in non-irradiated control skin up to two centimeters from the irradiation site. All the lesions persist for at least two days post-irradiation. These results sustain the hypothesis that pyrimidine dimer formation and excision could be a modality of epidermal communication.

Transcription coupled nucleotide excision repair by isolated Escherichia coli membrane-associated nucleoids.

One form of nucleotide excision repair (NER) is known to be functionally coupled to transcription, but the nature of this functional link in Escherichia coli is still unclear. Here we have employed the isolated membrane-associated nucleoids from E.coli to examine this issue. We show that the isolated nucleoid fraction is capable of excision of UV-induced pyrimidine dimers when reconstituted with a cytoplasmic fraction resolved by sucrose gradient fractionation. This excision activity by UvrABC is sensitive to rifampicin and is dependent on transcription. By using crosslinking and immunoprecipitation, the damage recognition protein, UvrA, was found to be specifically associated with the RNA polymerase beta subunit on the chromosomal DNA independent of DNA damage. It suggests that at least in one of the NER pathways the search for damage may be directly linked to RNA polymerase. In addition, the role of transcription in the unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA is described. This study provides insight into the understanding of the transcription-repair coupling in vivo.

Repair of cyclobutane pyrimidine dimers in unstimulated human mononuclear cells is deficient at very low fluences of ultraviolet B and is not enhanced by addition of deoxyribonucleosides.

Unstimulated human T lymphocytes are exquisitely sensitive to UVB irradiation. This hypersensitivity appears to relate to low deoxyribonucleotide pool sizes. They have also been reported to be defective in global excision of cyclobutane pyrimidine dimers, but such experiments may have been carried out at supralethal doses, where unrepaired excision breaks persist indefinitely. We use a T4 endonuclease Comet assay to show that removal of cyclobutane pyrimidine dimers is defective in the unstimulated mononuclear cell fraction (mainly T lymphocytes) even at sublethal fluences from an FS20 broad spectrum UVB lamp. Moreover, removal is not enhanced by addition of deoxyribonucleosides to the medium. Cells which are failing to remove cyclobutane pyrimidine dimers readily form fresh incision breaks in response to a second UVB fluence, indicating that they retain repair capacity and suggesting that removal of types of damage other than cyclobutane pyrimidine dimers is effective.

The effect of the OmpT protease on excision repair in UV-irradiated Escherichia coli

The extent of pyrimidine dimer excision (PDE) was inhibited in UV-irradiated E. coli KS272 ( ompT′) cells when they were preinduced by a low UV predose preceeded by a nutrition stress but not in the preinduced E. coli SF100 ( ompT) mutants. The preinduction, however, markedly inhibited PDE in the ompT cells transformed with a multicopy plasmid carrying ompT gene. The data are consistent with the hypothesis that the inducible OmpT protease (controlled by rpoH) might terminate the SOS period of excision repair so that when cells are preinduced PDE might be inhibited prematurely.

The pre-UV nutritional stresses increase UV resistance, decrease UV mutagenesis and inhibit excision repair

Nutritional stresses applied to E. coli prior to UV irradiation increase UV resistance and decrease UV mutagenesis. This effect is uvrA-dependent and might reflect a more efficient excision of pyrimidine dimers [1]. The data presented here, however, indicate that after prestarvation for glucose or amino acids pyrimidine dimer excision (PDE) was partly inhibited. It appears that the stress conditions stimulate a mode of uvr-dependent tolerance of lesions, efficient and precise. Possible modes of PDE inhibition and lesion tolerance are discussed.

The fission yeast UVDR DNA repair pathway is inducible.

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.

Mechanism of pyrimidine dimer excision repair: structural basis of damaged DNA recognition

Mechanism of pyrimidine dimer excision repair: structural basis of damaged DNA recognition

Mutagenesis and ultraviolet inactivation of transforming DNA of Haemophilus influenzae complexed with a Bacillus subtilis protein that alters DNA conformation.

The wild-type Bacillus subtilis spore protein, SspCwt, binds to DNA in vitro and in vivo and changes the conformation of DNA from B to A. Synthesis of the cloned SspCwt gene in Escherichia coli also causes large increases in mutation frequency. Binding of SspCwt to transforming DNA from Haemophilus influenzae made the DNA resistant to ultraviolet (UV) radiation. The mutant protein, SspCala, which does not bind DNA, did not change the UV resistance. The UV sensitivity of the DNA/SspCwt complex was not increased when the recipients of the DNA were defective in excision of pyrimidine dimers. These data indicate that the H. influenzae excision mechanism does not operate on the spore photoproduct formed by UV irradiation of the complex. Selection for the streptomycin- or erythromycin-resistance markers on the transforming DNA evidenced significant mutations at loci closely linked to these, but not at other loci. SspCwt apparently entered the cell attached to the transforming DNA, and caused mutations in adjacent loci. The amount of such mutations decreased when the transforming DNA was UV irradiated, because UV unlinks linked markers.

Constitutive increase of RecA protein: its influence on pyrimidine dimer excision and survival of UV-irradiated Escherichia coli.

Transformation of E. coli with the plasmid pRA containing recA gene increased the constitutive level of RecA protein 50-67 fold. This slightly inhibited pyrimidine dimer excision and reduced cell survival in three investigated, UV-irradiated E. coli strains. Our data support the view that RecA protein prematurely present at a high level may mask the dimers. The masking subsequently reduces the dimer excision and switches off the inducing signal.

Chromatid exchanges may be induced by damage in sites of transcriptional activity.

A conditional expression system has allowed us to vary the expression level of the xeroderma pigmentosum group A (XPA) photoproduct-specific DNA-binding protein in human cells and so control the response of cells to damage by UV light. Using a form of XPA that contains a single missense mutation (R207G) enabled us to study a lower range of function than that obtained with the wild-type sequence. This form of XPA has been previously shown to stimulate pyrimidine dimer excision preferentially in actively transcribed genes. We found that UV resistance increased as a linear function of XPA expression levels. Excision of (6-4) pyrimidine-pyrimidone photoproducts in the whole genome increased to a maximum at about the haploid level of XPA expression, but there was little pyrimidine dimer excision from the whole genome. SCE frequency induced by UV light was high in cells with no SPA expression and fell rapidly with increasing levels of SPA expression within 0-50% of the haploid level of expression. No further reduction in SCE frequency was produced at the highest levels of XPA expression, when repair replication extended to the overall genome. We speculate that a low level of repair, especially that occurring in actively transcribed genes, may selectively eliminate photoproducts that are particularly important in causing cell killing and SCEs.

A novel DNA N-glycosylase activity of E. coli T4 endonuclease V that excises 4,6-diamino-5-formamidopyrimidine from DNA, a UV-radiation- and hydroxyl radical-induced product of adenine.

We report on a novel activity of T4 endonuclease V. This enzyme is well known to be specific for the excision of pyrimidine dimers from UV-irradiated DNA. In this work, we show that T4 endonuclease V excises 4,6-diamino-5-formamidopyrimidine from DNA. 4,6-Diamino-5-formamidopyrimidine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation. DNA substrates were prepared by UV-or gamma-irradiation of DNA in aqueous solution. DNA substrates were incubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme. After incubation, DNA was precipitated and supernatant fractions were separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results provide evidence for the excision of 4,6-diamino-5-formamidopyrimidine by T4 endonuclease V from both gamma-and UV-irradiated DNA. Kinetics of excision were also determined. Fifteen other pyrimidine- and purine-derived base lesions that were identified in DNA samples were not substrates for this enzyme. It was concluded that, in addition to its well known activity for pyrimidine photodimers, T4 endonuclease V possesses an N-glycosylase activity for a major UV-radiation- and hydroxyl radical-induced monomeric product in DNA.

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a dna substrate: Structural basis for damaged DNA recognition

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 Å resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60° inclination at the central thymine dimer. The adenine base complementary to the 5′ side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.