Activation of p70 ribosomal protein S6 kinase is an essential step in the DNA damage-dependent signaling pathway responsible for the ultraviolet B-mediated increase in interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts.
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Ultraviolet B (UVB) irradiation has been shown to stimulate the expression of matrix-degrading metalloproteinases via generation of DNA damage and/or reactive oxygen species. Matrix-degrading metalloproteinases promote UVB-triggered detrimental long term effects like cancer formation and premature skin aging. Here, we were interested in identifying components of the signal transduction pathway that causally link UVB-mediated DNA damage and induction of matrix-degrading metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in human dermal fibroblasts in vitro. The activity of p70 ribosomal S6 kinase, a downstream target of the FK506-binding protein-12/rapamycin-associated protein kinase (FRAP) kinase (RAFT1, mTOR), was identified to be 4.8 +/- 0.8-fold, and MMP-1 and MMP-3 protein levels 2.4- and 11.5-fold increased upon UVB irradiation compared with mock-irradiated controls. The FRAP kinase inhibitor rapamycin and the DNA repair inhibitor aphidicolin significantly suppressed the UVB-mediated increase in p70 ribosomal S6 kinase activity by 50-65% and MMP-1 and MMP-3 protein levels by 34-68% and 42-88% compared with UVB-irradiated fibroblasts. By contrast, the interleukin-1beta-mediated increase in MMP-1 and MMP-3 protein levels could not be suppressed by rapamycin. Collectively, our data suggest that the FRAP-controlled p70 ribosomal S6 kinase is an essential component of a DNA damage-dependent, but not of the interleukin-1/cell membrane receptor-dependent signaling. (+info)
Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin.
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Ultraviolet-B (UVB) (290-320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40-45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection. (+info)
The xeroderma pigmentosum group C gene leads to selective repair of cyclobutane pyrimidine dimers rather than 6-4 photoproducts.
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We investigated the contribution of the xeroderma pigmentosum group C (XPC) gene to DNA repair. We stably transfected XPC cells (XP4PA-SV-EB) with XPC cDNA and selected a partially corrected (XP4PA-SE1) and a fully corrected (XP4PA-SE2) clone. Cell survival after UVC (254 nm) exposure was low for XP4PA-SV-EB, intermediate for XP4PA-SE1, and normal for XP4PA-SE2 cells. XP4PA-SV-EB cells had undetectable XPC mRNA and protein levels. XP4PA-SE1 cells had 130% of normal mRNA but 25% of normal protein levels, whereas XP4PA-SE2 cells had an 18-fold mRNA overexpression and normal XPC protein levels compared with normal cells. We measured cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) by using specific mAbs and the ELISA technique. XP4PA-SV-EB cells had no detectable removal of CPD or 6-4PP from their global genome by 24 h after 30 J/m(2) UVC exposure. The partially corrected XP4PA-SE1 cells had normal repair of CPD but minimal repair of 6-4PP by 24 h, whereas the fully corrected XP4PA-SE2 cells regained normal CPD and 6-4PP repair capacities. We also exposed pRSVcat plasmid to UVC (to induce CPD and 6-4PP), to UVC + photolyase (to leave only 6-4PP on the plasmid), or to UVB + acetophenone (to induce only CPD). Host cell reactivation of UVB + acetophenone-, but not of UVC + photolyase-treated plasmids was normal in XP4PA-SE1 cells. Thus, increasing XPC gene expression leads to selective repair of CPD in the global genome. Undetectable XPC protein is associated with no repair of CPD or 6-4PP, detectable but subnormal XPC protein levels reconstitute CPD but not 6-4PP repair, and normal XPC protein levels fully reconstitute both CPD and 6-4PP repair. (+info)
Fidelity of human DNA polymerase eta.
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Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer. (+info)
Specificity of DNA lesion bypass by the yeast DNA polymerase eta.
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DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer. We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol(eta) lacked a nuclease or a proofreading activity. It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively. The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases. However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion. These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone. (+info)
The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA.
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HBsu, the Bacillus subtilis homolog of the Escherichia coli HU proteins and the major chromosomal protein in vegetative cells of B. subtilis, is present at similar levels in vegetative cells and spores ( approximately 5 x 10(4) monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the alpha/beta-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with alpha/beta-type SASP on the nucleoid, suggesting that HBsu could modulate alpha/beta-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered alpha/beta-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing alpha/beta-type SASP-mediated positive supercoiling, and greatly ameliorated the alpha/beta-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the alpha/beta-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of alpha/beta-type SASP on the properties of DNA in the developing and dormant spore. (+info)
Cutaneous melanoma patients have normal repair kinetics of ultraviolet-induced DNA repair in skin in situ.
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The DNA lesions induced by ultraviolet radiation include cyclobutane pyrimidine dimers and 6-4 photoproducts. We investigated whether cutaneous melanoma patients have an impaired ability to repair their ultraviolet-induced photolesions. Seventeen patients with melanoma and 13 healthy controls took part in this study. Both groups received a dose of 40 mJ per cm2 Commission Internationale de l'Eclairage of solar simulating radiation on previously unexposed buttock skin. Skin biopsies were taken at 0 h, 24 h, and 48 h after ultraviolet exposure. A 32P-postlabeling method was used to measure both cyclobutane pyrimidine dimers and 6-4 photoproducts in skin. Cyclobutane pyrimidine dimers and 6-4 photoproduct levels did not differ in the melanoma patients from those in the control group at any time point post-ultraviolet radiation. The repair rate of cyclobutane dimer TT=C was faster than that for TT=T both at 24 h and 48 h postirradiation in both groups, providing evidence of site-specific repair (p < 0.05). We conclude that patients with melanoma have a normal ultraviolet-induced DNA repair capacity in skin in situ. (+info)
Solution structure of the DNA decamer duplex containing a 3'-T x T basepair of the cis-syn cyclobutane pyrimidine dimer: implication for the mutagenic property of the cis-syn dimer.
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The cis - syn dimer is the major DNA photoproduct produced by UV irradiation. In order to determine the origin of the mutagenic property of the cis - syn dimer, we used NMR restraints and molecular dynamics to determine the solution structure of a DNA decamer duplex containing a wobble pair between the 3'-T of the cis - syn dimer and the opposite T residue (CS/TA duplex). The solution structure of the CS/TA duplex revealed that the 3'-T x T base pair of the cis - syn dimer had base pair geometry that was significantly different from the canonical Watson-Crick base pair and caused destabilization and conformational distortion of its 3'-region. However, a 3'-T x A base pair at the cis - syn dimer within this related DNA decamer maintains the normal Watson-Crick base pair geometry and causes little distortion in the conformation of its 3'-side. Our results show that in spite of its stable hydrogen bonding, the insertion of a T residue opposite the 3'-T of the cis - syn dimer is inhibited by structural distortion caused by the 3'-T x T base pair. This may explain why the frequency of the 3'-T-->A transversion, which is the major mutation produced by the cis - syn dimer, is only 4%. (+info)