In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.

The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes

Chromatin Remodeling Activities Act on UV-damaged Nucleosomes and Modulate DNA Damage Accessibility to Photolyase

Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome.

The organization of DNA within eukaryotic cell nuclei poses special problems and opportunities for the cell. For example, assembly of DNA into chromatin is thought to be a principle mechanism by which adventitious general transcription is repressed. However, access to genomic DNA for events such as DNA repair must be facilitated by energy-intensive processes that either directly alter chromatin structure or impart post-translational modifications, leading to increased DNA accessibility. The assembly of DNA into chromatin affects both the incidence of damage to DNA and repair of that damage. Correction of most damage to DNA caused by UV irradiation occurs via the nucleotide excision repair (NER) process. NER requires extensive involvement of large multiprotein complexes with relatively large stretches of DNA. Here, we review recent evidence suggesting that at least some steps of NER require ATP-dependent chromatin remodeling activities while perhaps others do not.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1033.2002.02888.x

Nucleotide excision repair and chromatin remodeling.

The kinetics of thymine–thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine–thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5′-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of nearly 1000 bases made it possible to monitor damage at all 9 tetrads of the type XTTY, where X and Y are non-thymine bases. TT-CPD yields for different tetrads were found to vary by as much as an order of magnitude, but similar yields were observed at all instances of a given tetrad. Kinetic analysis of CPD formation at 23 distinct sites reveals that both the formation and reversal photoreactions depend sensitively on the identity of the nearest-neighbour bases on the 5′ and the 3′ side of a photoreactive TT base step. The lowest formation and reversal rates occur when two purine bases flank a TT step, while the highest formation and reversal rates are observed for tetrads with at least one flanking C. Overall, the results show that the probabilities of CPD formation and photoreversal depend principally on interactions with nearest-neighbour bases.

/pdf/sequence-dependent-thymine-dimer-formation-and-photoreversal-2snlvxxr8q.pdf

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of themore » CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.« less

Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.

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Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.