The carboxy-terminal domain of the XPC protein plays a crucial role in nucleotide excision repair through interactions with transcription factor IIH.
ABSTRACT The xeroderma pigmentosum group C (XPC) protein specifically involved in genome-wide damage recognition for nucleotide excision repair (NER) was purified as a tight complex with HR23B, one of the two mammalian homologs of RAD23 in budding yeast. This XPC-HR23B complex exhibits strong binding affinity for single-stranded DNA, as well as preferential binding to various types of damaged DNA. To examine the structure-function relationship of XPC, a series of truncated mutant proteins were generated and assayed for various binding activities. The two domains participating in binding to HR23B and damaged DNA, respectively, were mapped within the carboxy-terminal half of XPC, which also contains an evolutionary conserved amino acid sequence homologous to the yeast RAD4 protein. We established that the carboxy-terminal 125 amino acids are dispensable for both HR23B and damaged DNA binding, while interactions with transcription factor IIH (TFIIH) are significantly impaired by truncation of this domain. Furthermore, deletion of the extreme carboxy-terminal domain totally abolished XPC activity in the cell-free NER reaction. These results suggest that following initial damage recognition, the carboxy terminus of XPC may be essential for the recruitment of TFIIH, and that most truncation mutations identified in XP-C patients result in non-functional proteins.
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ABSTRACT: Cellular mismatch and base-excision repair machineries have been shown to be involved in Epstein-Barr Virus (EBV) lytic DNA replication. We report here that nucleotide-excision repair (NER) may also play an important role in EBV lytic DNA replication. Firstly, the EBV BGLF4 kinase interacts with xeroderma pigmentosum C (XPC), the critical DNA damage-recognition factor of NER, in yeast and in vitro, as demonstrated by yeast two-hybrid and glutathione S-transferase pull-down assays. Simultaneously, XPC was shown, by indirect immunofluorescence and co-immunoprecipitation assays, to interact and colocalize with BGLF4 in EBV-positive NA cells undergoing lytic viral replication. In addition, the efficiency of EBV DNA replication was reduced about 30-40 % by an XPC small interfering RNA. Expression of BGLF4 enhances cellular DNA-repair activity in p53-defective H1299/bcl2 cells in a host-cell reactivation assay. This enhancement was not observed in the XPC-mutant cell line XP4PA-SV unless complemented by ectopic XPC, suggesting that BGLF4 may stimulate DNA repair in an XPC-dependent manner. Overall, we suggest that the interaction of BGLF4 and XPC may be involved in DNA replication and repair and thereby enhance the efficiency of viral DNA replication.Journal of General Virology 01/2008; 88(Pt 12):3234-43. DOI:10.1099/vir.0.83212-0 · 3.53 Impact Factor
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ABSTRACT: Arabidopsis thaliana CENTRIN2 (AtCEN2) has been shown to modulate Nucleotide Excision Repair (NER) and Homologous Recombination (HR). The present study provides evidence that AtCEN2 interacts with the Arabidopsis homolog of human XPC, AtRAD4 and that the distal EF-hand Ca(2+) binding domain is essential for this interaction. In addition, the synthesis-dependent repair efficiency of bulky DNA lesions was enhanced in cell extracts prepared from Arabidopsis plants overexpressing the full length AtCEN2 but not in those overexpressing a truncated AtCEN2 form, suggesting a role for the distal EF-hand Ca(2+) binding domain in the early step of the NER process. Upon UV-C treatment the AtCEN2 protein was shown to be increased in concentration and to be localised in the nucleus rapidly. Taken together these data suggest that AtCEN2 is a part of the AtRAD4 recognition complex and that this interaction is required for efficient NER. In addition, NER and HR appear to be differentially modulated upon exposure of plants to DNA damaging agents. This suggests in plants, that processing of bulky DNA lesions highly depends on the excision repair efficiency, especially the recognition step, thus influencing the recombinational repair pathway.Plant Molecular Biology 06/2006; 61(1-2):345-56. DOI:10.1007/s11103-006-0016-9 · 4.07 Impact Factor
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ABSTRACT: Mutations that extend lifespan in invertebrates often lead to resistance to multiple forms of stress, suggesting that stress resistance might also be important in lifespan determination in mammals. Work in this thesis supports this idea from several perspectives. Snell dwarf mice have a mutation in the gene Pit1 which extends their lifespan approximately 40% relative to controls and delays the progression of multiple age-related pathologies. We found that skin-derived fibroblast cultures grown from Snell dwarf mice are resistant to cell death induced by both oxidative stresses and stresses that are, in part, oxidation-independent, like heat, heavy metals, and DNA damaging agents. We also found that fibroblast resistance to the oxidative stressor peroxide is correlated with resistance to other stressors, both oxidative and non-oxidative, suggesting regulation of these properties by an overlapping set of mechanisms. We found similar patterns of resistance in fibroblasts grown from mice with other lifespan-extending mutations. An additional set of experiments showed that Snell dwarf fibroblasts have an enhanced ability to repair UV-induced DNA lesions, providing a possible explanation for their resistance to death cause by UV irradiation. Snell dwarf fibroblasts express higher levels of two nucleotide excision proteins than control, which may contribute to their enhanced DNA repair and to delayed aging and diminished neoplasia in these mice. Extending these approaches to comparative biology, we found that resistance to some cellular stressors is correlated with maximum lifespan across mammalian species. Fibroblasts from long-lived species of rodents are significantly resistant to heavy metals and some oxidative stressors, and show similar trends for death caused by heat or DNA alkylation. For other agents however, such as UV light, there was no association between lifespan and cellular stress resistance. These results suggest that as rodent species evolved longer lifespan, there was also a coordinate increase in the cellular resistance to many, but not all, cellular stressors. Overall, these results support the idea that mechanisms that regulate lifespan in mammals also tend to increase cellular stress resistance. The further study of the mechanisms of stress resistance in mammals may then help us better understand the molecular regulation of aging. Ph.D. Cellular & Molecular Biology University of Michigan, Horace H. Rackham School of Graduate Studies http://deepblue.lib.umich.edu/bitstream/2027.42/57719/2/asalmon_1.pdf