Hamster Cells Are Deficient in UV-DDB
UV-DDB is absent or expressed at very low levels in 10 Chinese hamster cell lines (Hwang et al., 1998
). To determine whether the low level of UV-DDB was an artifact of cell culture or a property shared by primary hamster tissues, we measured UV-DDB in extracts of peripheral blood lymphocytes isolated directly from Syrian golden hamsters.
Hamster lymphocytes contained barely detectable levels of UV-DDB (, lanes 5 and 6), while extracts from human peripheral blood lymphocytes contained at least 30-fold higher levels of UV-DDB (lanes 2 and 3). Similarly, UV-DDB was expressed at barely detectable levels in hamster V79 cells (lanes 9 and 10), but expressed at high levels in human HeLa cells (lanes 7 and 8). Low levels of UV-DDB were also seen in primary mouse tissues, including peripheral blood lymphocytes, spleen, heart, lung, kidney, and skin (data not shown). The lack of UV-DDB in rodent cells is not due to absence of the p48
gene, since treatment of hamster cells with the demethylating agent azacytidine induces expression of UV-DDB (Hwang et al., 1998
). Thus, UV-DDB may be transcriptionally suppressed in many rodent tissues.
p48 Confers UV-DDB to Hamster Cells
Hamster Cell Lines Transfected with p48 Express UV-DDB
The failure of hamster cells to express UV-DDB presented an opportunity to define its role in DNA repair, since hamster cell lines are highly receptive to DNA transfection. An expression vector lacking a cDNA insert or containing either p48 or FLAG-p48 was transfected into V79 hamster cells, and clones were screened for UV-DDB by EMSA (). Little UV-DDB was detected in the parental V79 cell line or in V79 cells stably transfected with the control vector. However, clones with p48 message or FLAG-p48 protein have significant levels of UV-DDB (). Thus, expression of p48 conferred UV-DDB to the hamster cells.
Three clones were selected for further analysis: 1A, 3B4, and 5E. Expression of p48 had no significant effect on the division times of these clones: 16 hr for 1A (vec), 15 hr for 3B4 (p48), and 16 hr for 5E (FLAG-p48).
p48 Is Required for Global Genomic Repair of CPDs but Not 6–4 Photoproducts
To test the effect of p48 on GGR of 6–4 photoproducts, unreplicated DNA from UV-irradiated cells was probed with a monoclonal antibody against 6–4 photoproducts. Expression of p48 in hamster cells had no effect on the already rapid repair of 6–4 photoproducts ().
p48 Enhances Global Genomic Repair of CPDs
To test the effect of p48 on GGR of CPDs, unreplicated DNA from UV-irradiated cells was probed with a monoclonal antibody against CPDs. GGR of CPDs was undetectable in the hamster clone transfected with control vector, consistent with previous reports that hamster cell lines are defective in GGR of CPDs (Bohr et al., 1985
), but occurred at significant levels in hamster clones expressing FLAG-p48 or p48 ().
p48 Is Not Required for Transcription Coupled Repair of CPDs
To determine whether p48 has a role in TCR, we measured CPD repair in the transcribed and nontranscribed strands of the DHFR
gene. Wild-type hamster cells (wt) and hamster cells transfected with control vector showed negligible levels of CPD repair in the nontranscribed strand () as previously reported (Mellon et al., 1987
). Hamster cells expressing p48 showed a significantly higher level of CPD repair on the nontranscribed strand, consistent with the role of p48 in GGR. Expression of p48 had no effect on the proficient repair of CPDs from the transcribed strand.
p48 Expression Does Not Affect UV Survival
To determine the physiological significance of GGR of CPDs, we measured the effect of p48 on UV survival. Colony formation after UV was indistinguishable among wild-type hamster cells (V79, AA8) and hamster cells transfected with p48 or control vector (), demonstrating that GGR of CPDs does not affect UV survival. By contrast, decreased colony survival was observed in mutant cell lines with defects in the Cockayne syndrome B gene (UV61) and the XP group D gene (UV5) as previously reported (Friedberg et al., 1995
p48 Expression Does Not Affect UV Survival but Suppresses Mutagenesis
p48 Suppresses UV-Induced Mutagenesis
The V79 cell line was derived from a male hamster and contains one copy of the X-linked HPRT
ygene. In V79 cells transfected with vector, the spontaneous mutation rate in the HPRT
gene was very low, 0.7 mutations per 105
cells. When asynchronously growing cells were exposed to UV, the mutation rate increased to 15 per 105
cells at a dose of 2 J/m2
and 55 per 105
cells at a dose of 10 J/m2
(). Similar mutation rates (6.2 per 105
cells per J/m2
) were reported previously for V79 cells (Zdzienicka et al., 1988
Hamster cells expressing p48 also had a low spontaneous mutation rate, 1 per 105 cells. When the cells expressing p48 or FLAG-p48 were exposed to UV, the mutation rate was 7.5 per 105 cells at a dose of 2 J/m2 and 20 per 105 cells at a dose of 10 J/m2. Thus, expression of p48 in hamster cells suppressed UV-induced mutagenesis 2- to 2.7-fold. Suppression of mutagenesis was similar in hamster cells growing asynchronously or synchronized in G1 at the time of UV exposure.
p48 Suppresses Mutations Only from Nontranscribed DNA
Hamster cells were exposed to UV (2 J/m2
) and grown in divided populations to select for independent HPRT
mutant clones. lists independent mutations arising in the hamster cells transfected with vector or p48. Most mutations were single base pair substitutions, 29% of which were C-to-T transitions. Significantly, 3 tandem base pair mutations at dipyrimidines were observed: a CT-to-TC transition, a CC-to-TT transition, and a TT-to-AA transversion. Such mutations have been reported almost exclusively in cells from UV-induced skin tumors, and very rarely for internal tumors (Giglia et al., 1998
UV-Induced HPRT Mutations from Hamster Cells Transfected with Vector or p48
The damaged DNA strand that led to each HPRT mutation could be inferred in most cases from sequence context. For hamster cells transfected with vector, the vast majority of UV-induced lesions (94%) occurred at sites containing adjacent pyrimidines, where CPDs and 6–4 photoproducts could have formed. Only 22% (7 of 32) of the mutations were attributable to dipyrimidine lesions on the transcribed strand, while 72% (23 of 32) were from the nontranscribed strand (), consistent with the poor GGR of CPDs in hamster cells. By contrast, for hamster cells expressing p48, 36% (13 of 36) of the mutations were attributable to the transcribed strand, while 42% (15 of 36) were from the nontran-scribed strand. The effect of p48 on the strand specificity of mutations was statistically significant (p = 0.041 by Fisher’s exact test).
To calculate mutation rates for transcribed and non-transcribed DNA, the percentage of mutations found on each strand was multiplied by the overall mutation rate for the cell line (). Expression of p48 did not strongly affect the mutation rate on the transcribed strand, but decreased the mutation rate 3.7-fold on the nontranscribed strand, consistent with the role of p48 in repairing nontranscribed DNA.
p48 Suppresses Mutations Arising from Pyrimidine Dimers in the Nontranscribed Strand of DNA
UV induces CPDs at both TT and non-TT (CC, CT, or TC) dipyrimidine sites. In hamster cells transfected with control vector, more mutations arose from non-TT than from TT dipyrimidines (). This bias was seen on the transcribed strand (1 mutation from TT, 6 from non-TT) and the nontranscribed strand (7 from TT, 12 from non-TT). When p48 was expressed in hamster cells, there was no significant effect on mutations from the transcribed strand (3 from TT, 10 from non-TT). However, on the nontranscribed strand, the mutations from non-TT dipyrimidines declined significantly (9 from TT, 3 from non-TT). The difference in the effect of p48 on mutations arising from TT and non-TT dipyrimidines was statistically significant (p = 0.037 by Fisher’s exact test), suggesting that UV-DDB targets non-TT CPDs for repair more efficiently than TT CPDs ().