Tsukasa Matsunaga

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6‐4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET‐IRRADIATED DNA

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 photo‐reactivation, 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.

WAVELENGTH DEPENDENT FORMATION OF THYMINE DIMERS AND (6-4)PHOTOPRODUCTS IN DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT RANGING FROM 150 TO 365 nm

Abstract—We investigated the wavelength dependence of cyclobutane thymine dimer and (6‐4)photo‐product 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 with254–365 nm monochromatic UV from a spectrograph, or with220–300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6‐4)photoproducts were fluence‐dependently induced by every UV wavelength tested so far. We also examined the induction of both types of DNA damage by 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.

Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells

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.

Postnatal Growth Failure, Short Life Span, and Early Onset of Cellular Senescence and Subsequent Immortalization in Mice Lacking the Xeroderma Pigmentosum Group G Gene

ABSTRACT The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3′ nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the humanXPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. SinceXPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from thexpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

The detection of cyclobutane thymine dimers, (6-4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects

Ultraviolet irradiation of skin induces various DNA photolesions. Here we demonstrate that irradiation of human buttock skin with 300 nm UVR in situ induces thymine dimers and 6-4 photoproducts. Irradiation with 260 nm immediately followed by UVA (320 nm) induces the Dewar photoisomers of the 6-4 lesions. All three lesions can be detected in methanol-fixed paraffin sections using specific monoclonal antibodies. The sections have been analysed in an automated image analysis system (Discovery) and the level of immuno-DAB-peroxidase measured in individual epidermal cell nuclei as absorption at 460 nm (integrated optical density). The staining patterns with the antibodies showed no detectable change with epidermal depth by eye after 300 nm irradiation, however, the machine detected a fall off with depth of about 2.5% per cell layer. Following irradiation with a shorter wavelength (260 nm) there was a rapid fall off in staining with depth easily detectable by eye and machine (39% per cell layer).

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts.

We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.

Isolation of XAB2 complex involved in pre-mRNA splicing, transcription, and transcription-coupled repair

Nucleotide excision repair is a versatile repair pathway that counteracts the deleterious effects of various DNA lesions. In nucleotide excision repair, there is a transcription-coupled repair (TCR) pathway that focuses on DNA damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPA-binding protein 2), containing tetratricopeptide repeats, has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 has been shown to interact with Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and is involved in TCR and transcription. Here we purified XAB2 as a multimeric protein complex consisting of hAquarius, XAB2, hPRP19, CCDC16, hISY1, and PPIE, which are involved in pre-mRNA splicing. Knockdown of XAB2 with small interfering RNA in HeLa cells resulted in a hypersensitivity to killing by UV light and a decreased recovery of RNA synthesis after UV irradiation and regular RNA synthesis. Enhanced interaction of XAB2 with RNA polymerase IIo or XPA was observed in cells treated with DNA-damaging agents, indicating DNA damage-responsive activity of the XAB2 complex. These results indicated that the XAB2 complex is a multifunctional factor involved in pre-mRNA splicing, transcription, and TCR.

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV‐induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV‐irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M‐2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M‐2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V‐B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.

Ultraviolet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers

Patients with ultraviolet-sensitive syndrome (UV(S)S) are sensitive to sunlight, but present neither developmental nor neurological deficiencies. Complementation studies with hereditary DNA repair syndromes show that UV(S)S is distinct from all known xeroderma pigmentosum (XP) and Cockayne syndrome (CS) groups. UV(S)S cells exhibit some characteristics typical of CS, including normal global genomic (GGR) repair of UV-photoproducts, poor clonal survival and defective recovery of RNA synthesis after UV exposure. Those observations have led us to suggest that UV(S)S cells, like those from CS, are defective in transcription-coupled repair (TCR) of cyclobutane pyrimidine dimers (CPD). We have now examined the repair of CPD in the transcribed and non-transcribed strands of the active dihydrofolate reductase (DHFR) and p53 genes, and of the silent alpha-fetoprotein (AFP) and mid-size neurofilament (NF-M) genes in normal human cells and in cells belonging to UV(S)S and CS complementation group B. Our results provide compelling evidence that the UV(S)S gene is essential for TCR of CPD and probably other bulky DNA lesions. As a possible distinction between UV(S)S and CS patients, we postulate that the UV(S)S gene may not be required for TCR of oxidative lesions. We have also found that repair of CPD in either DNA strand of the genomic fragments examined, occurs at a slower rate in TCR-deficient cells than in the non-transcribed strands in normal cells; we suggest that in the absence of TCR, global repair complexes have hindered access to lesions in genomic regions that extend beyond individual transcription units.

Base sequence specificity of a monoclonal antibody binding to (6-4)photoproducts

The DNA base sequence specificity of the 64M-1 monoclonal antibody, which recognizes ultraviolet (UV)-induced (6-4)photoproducts, was characterized. The 64M-1 antibody strongly bound to UV-poly(dU) as well as to UV-poly(dT), and weakly to UV-poly(dC), UV-poly(me5dC) and UV-poly(rU). A competitive inhibition assay using UV-oligo(dT)8, UV-oligo(dTdC)4, UV-oligo(dC)8, UV-PvuI linker (GCGATCGC) and UV-PvuII linker (GCAGCTGC) indicated that the main (6-4)photoproducts detected by the 64M-1 antibody in UV-irradiated DNA are TT(6-4)photoproducts and TC(6-4)photoproducts. Comparison between dTpdT(6-4)photoproduct and dTpdC(6-4)photoproduct showed that the affinity of the 64M-1 antibody for dTpdT(6-4)photoproduct was about 5 times higher than that for dTpdC(6-4)photoproduct. The antibody also binds to isolated TT(6-4)photoproducts.