Immunohistochemical Analysis of Nucleotide Excision Repair Factors XPA and XPB in Adult Rat Brain

To study the regional and cellular distribution of xeroderma pigmentosum group A and B (XPA and XPB) proteins, two nucleotide excision repair (NER) factors, in the mammalian brain we used immunohistochemistry and triple fluorescent immunostaining combined with confocal microscope scanning in brain slices of adult rat brain, including the cerebral cortex, striatum, substantia nigra compacta, ventral tegmental area, red nucleus, hippocampus, and cerebellum. Both XPA and XPB proteins were mainly expressed in neurons, because the XPA‐ or XPB‐immunopositive cells were only costained with NeuN, a specific neuronal marker, but not with glial fibrillary acidic acid, a specific astrocyte marker, in the striatum. Furthermore, XPA‐ and XPB‐positive staining were observed in the neuronal nuclei. Such subcellular distribution was consistent with the location of the NER in the cells. This study provides the first evidence that NER factors XPA and XPB exist in the nuclei of neurons in the brain, suggesting that the NER may play important roles in the process of DNA repair in adult brain neurons. Anat Rec, 291:775–780, 2008. © 2008 Wiley‐Liss, Inc.     

Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress

Nucleotide excision repair (NER) is the most flexible of all known DNA‐repair mechanisms, and XPG is a 3′‐endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP‐G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP‐G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV‐irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV‐ but not oxidative‐stress‐induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.