No abstract

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)photoproducts) are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by ultraviolet light irradiation of cells. The phenomenon of photoreactivation, the reduction of the lethal and mutagenic effects of ultraviolet radiation by simultaneous or subsequent irradiation with near ultraviolet or visible light, has been identified in several organisms and in some cases the enzymes that catalyse this process have been characterized in sufficient detail. CPDs are the only known substrate for the photoreactivating enzymes so far analysed and enzymatic photoreactivation of (6-4)photoproducts has not yet been reported. We report here that an enzyme that catalyses the light-dependent repair of (6-4)photoproduct exists in Drosophila melanogaster. This is, to our knowledge, the first report of such photoreactivating activity specific for (6-4)photoproducts in any organism.

Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by a high frequency of skin cancer on sun-exposed areas, and neurological complications. XP has a defect in the early step(s) of nucleotide-excision repair (NER) and consists of eight different genetic complementation groups (groups A-G and a variant). We established XPA (group-A XP) gene-deficient mice by gene targeting of mouse embryonic stem (ES) cells. The XPA-deficient mice showed neither obvious physical abnormalities nor pathological alterations, but were defective in NER and highly susceptible to ultraviolet-B- or 9,10-dimethyl-1,2-benz[a]anthracene-induced skin carcinogenesis. These findings provide in vivo evidence that the XPA protein protects mice from carcinogenesis initiated by ultraviolet or chemical carcinogen. The XPA-deficient mice may provide a good in vivo model to study the high incidence of skin carcinogenesis in group A XP patients.

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC⅐HR23B, TFIIH, XPF⅐ERCC1 and XPG, up to 17-fold for CPDs and ϳ2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.Xeroderma pigmentosum (XP) 1 is a rare autosomal recessive disease characterized by sun sensitivity, pigmentation abnormalities, and high incidence of skin cancer (1, 2). XP is genetically heterogeneous and mutations in eight different genes (XPA through XPG and XPV) are known to cause this disease. All XP gene products, except XPV, are involved in nucleotide excision repair (NER), which removes a wide variety of DNA damages by dual incisions on both sides of the lesion (3-5). However, the function of damaged DNA-binding protein (DDB), which is linked to XP group E, is poorly understood.DDB was originally identified as a nuclear factor that binds to UV-damaged DNA and has been shown to recognize a wide spectrum of DNA lesions (6 -11). It is a heterodimer of p127 and p48, and both subunits are required for the activity (9, 12, 13). It has been reported that the mRNA levels of p48, but not of p127, strongly depend on the tumor suppressor p53 and increase further after DNA damage in a p53-dependent manner (14). Correspondingly, the protein levels and the activity of DDB also increase after UV irradiation (8,15). A subset of cell strains from XP group E patients were found to be deficient in the DDB activity (Ddb Ϫ XP-E) and to have mutations in p48 gene (7,12,16,17), although Ddb ϩ strains may have been misclassified as XP-E (18). The recent in vivo studies showed that XP-E cells are selectively defective in global genomic repair (GGR) (14), which repairs lesions from both nontranscribed genomic DNA and the nontranscribed strand of expressed genes. Chu and colleagues have demonstrated that Chinese hamster cells lack DDB activity...

We investigated the wavelength dependence of cyclobutane thymine dimer and (6-4)photoproduct induction by monochromatic UV in the region extending from 150 to 365 nm, using an enzyme-linked immunosorbent assay with two monoclonal antibodies. Calf thymus DNA solution was irradiated with 254-365 nm monochromatic UV from a spectrograph, or with 220-300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6-4)photoproducts were fluence-dependently induced by every UV below 220 nm extending to 150 nm under dry condition. We detected the efficient formation of both types of damage in the shorter UV region, as well as at 260 nm, which had been believed to be the most efficient wavelength for the formation of UV lesions. The action spectra for the induction of thymine dimers and (6-4)photoproducts were similar from 180 to 300 nm, whereas the action spectrum values for thymine dimer induction were about 9- and 1.4-fold or more higher than the values for (6-4)photoproduct induction below 160 nm and above 313 nm, respectively.

XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junctioncutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNAbinding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF-ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease.Nucleotide excision repair is a general repair system that plays an important role in maintaining genetic integrity (1, 2). This repair system removes damaged nucleotides from DNA by dual incisions on both sides of the lesion in the damaged strand (3). Recently, the human and the highly homologous yeast nucleotide excision repair systems have been reconstituted from extensively purified proteins, which demonstrated that 14 -15 polypeptides comprising replication protein A (RPA, 1 also known as HSSB or RF-A) and the general transcription factor, TFIIH, in addition to XP and ERCC proteins were essential for the dual incision step (4 -6).It was found that the two subunits of the excision nuclease had intrinsic endonuclease activities in the absence of the other components of the repair system. XPG was reported to possess a single-stranded DNA endonuclease activity (7,8) and an exonuclease activity with 5Ј to 3Ј directionality (9). Similarly, it was found that XPF-ERCC1 had a single strand-specific endonuclease activity and a weak activity on double-stranded UVirradiated DNA, which was stimulated by RPA (10). Furthermore, it has been reported that the RAD1-RAD10 complex (11), the yeast counterpart of the human XPF-ERCC1 nuclease, and XPG nuclease (12) have structure-specific junction-cutting activities with unique polarities. These results obtained with the model "undamaged" substrates suggested that XPF-ERCC1 and XPG were the 5Ј-and 3Ј-endonucleases of the dual incisions, respectively. A recent study using damaged DNA and specific antibodies supported this model (13).Recently, it has been reported that RPA, which is absolutely required for dual incisions (4), specifically binds to XPA protein and XPG nuclease (14). These findings raised the possibility that RPA may play an important role in recognizing DNA lesions and then targeting the nuclease subunits of hu...

Human histone H2AX is rapidly phosphorylated on serine 139 in response to DNA double-strand breaks and plays a crucial role in tethering the factors involved in DNA repair and damage signaling. Replication stress caused by hydroxyurea or UV also initiates H2AX phosphorylation in S-phase cells, although UV-induced H2AX phosphorylation in non-cycling cells has recently been observed. Here we study the UV-induced H2AX phosphorylation in human primary fibroblasts under growth-arrested conditions. This reaction absolutely depends on nucleotide excision repair (NER) and is mechanistically distinct from the replication stress-induced phosphorylation. The treatment of cytosine-β-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions. Consistently, the phosphorylation appears to be mainly mediated by ataxia-telangiectasia mutated and Rad3-related (ATR), although Chk1 (Ser345) is not phosphorylated by the activated ATR. The cellular levels of DNA polymerases δ and ϵ and proliferating cell nuclear antigen are markedly reduced in quiescent cells. We propose a model that perturbed gap-filling synthesis following dual incision in NER generates single-strand DNA gaps and hence initiates H2AX phosphorylation by ATR with the aid of RPA and ATRIP.