Volume 5, Issue 4, Pages (April 2000)

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Volume 5, Issue 4, Pages 737-744 (April 2000) Xeroderma Pigmentosum p48 Gene Enhances Global Genomic Repair and Suppresses UV-Induced Mutagenesis  Jean Y Tang, Byung Joon Hwang, James M Ford, Philip C Hanawalt, Gilbert Chu  Molecular Cell  Volume 5, Issue 4, Pages 737-744 (April 2000) DOI: 10.1016/S1097-2765(00)80252-X

Figure 1 p48 Confers UV-DDB to Hamster Cells (A) Deficiency of UV-DDB in hamster cells. UV-DDB was measured in human and hamster peripheral blood lymphocytes and human (HeLa) and hamster (V79) cell lines. Whole-cell extracts were incubated with a 148 bp 32P-labeled DNA probe, which was either left intact (−) or irradiated with 5000 J/m2 of UV (+), and resolved by electrophoretic mobility shift assay (EMSA) (Hwang et al. 1999). B1 and B2 mark the mobility of UV-DDB/DNA complexes. F marks the mobility of free DNA probe. (B) Expression of UV-DDB in cells transfected with p48. Hamster V79 cells were stably transfected with an expression vector lacking a cDNA insert (vec) or containing cDNA encoding p48 or FLAG-p48. Extracts (2 μg) were assessed for UV-DDB by EMSA. Levels of p48, p125, and actin mRNA were measured by Northern blot of total RNA (10 μg per lane), and levels of FLAG-p48 protein were measured by immunoblot with anti-FLAG antibodies (2 μg/ml) (Hwang et al. 1998). Molecular Cell 2000 5, 737-744DOI: (10.1016/S1097-2765(00)80252-X)

Figure 2 p48 Enhances Global Genomic Repair of CPDs (A) GGR of 6–4 photoproducts. (B) GGR of CPDs. Repair was measured after exposure to UV in hamster cells expressing p48, FLAG-p48, or vector. Each point represents the mean of three independent experiments. (C) TCR of CPDs. Removal of CPDs from the nontranscribed and transcribed strands of the DHFR gene was measured after exposure to UV. Molecular Cell 2000 5, 737-744DOI: (10.1016/S1097-2765(00)80252-X)

Figure 3 p48 Expression Does Not Affect UV Survival but Suppresses Mutagenesis (A) UV survival. Wild-type hamster cells (V79 or AA8), V79 hamster cells transfected with p48 or vector, and hamster cells with mutations in the Cockayne syndrome group B gene (CSB−) or the XP group D gene (XPD−) were exposed to UV from a Westinghouse IL782–30 germicidal lamp at an incident dose of 0.33 J/m2/s. Surviving colonies were counted after 2 weeks. (B) UV-induced mutagenesis. Mutations in the HPRT gene were measured in hamster cells stably transfected with p48, FLAG-p48, or vector. Cells were grown asynchronously (asyn) or synchronized in G1 (syn) by serum starvation, exposed to different doses of UV, and selected for HPRT mutations with 6-thioguanine. Molecular Cell 2000 5, 737-744DOI: (10.1016/S1097-2765(00)80252-X)

Figure 4 p48 Suppresses Mutations Arising from Pyrimidine Dimers in the Nontranscribed Strand of DNA (A) Strand bias. UV-induced mutations in the HPRT gene were measured in hamster cells stably transfected with vector or p48. The percentage of mutations on the transcribed strand (TS) and nontranscribed strand (NTS) was multiplied by the overall mutation rates to obtain strand-specific mutation rates. (B) TT versus non-TT bias. Mutation rates are shown for TT and non-TT dipyrimidine sequences. Cases in which the dipyrimidine could have been either TT or non-TT (Table 1) were all on the NTS, and these were not included in the analysis. Molecular Cell 2000 5, 737-744DOI: (10.1016/S1097-2765(00)80252-X)