The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.

In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.     

Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPD) and accelerates repair of 6-4 photoproducts (6-4PP). The high UV-induced skin cancer susceptibility of mice compared to man has been attributed to low expression of the UV-DDB subunit DDB2 in mouse skin cells. However, DDB2 knockout mice exhibit enhanced UVB skin carcinogenesis indicating that DDB2 protects mice against UV-induced skin cancer. To resolve these apparent contradictory findings, we systematically investigated the NER capacity of mouse fibroblasts and keratinocytes. Compared to fibroblasts, keratinocytes exhibited an increased level of UV-DDB activity, contained significantly higher levels of other NER proteins (i.e. XPC and XPB) and displayed efficient repair of CPD. At low UVB dosages, the difference in skin cancer susceptibility between DDB2 KO and wild type mice was even much more pronounced than previously reported with high dose UVB exposures. Hence, our observations show that mouse keratinocytes express sufficient levels of UV-DDB for efficient repair of photolesions and efficient protection against UV-induced skin cancer at physiological relevant UV exposure.     

Characterization of the rhp7(+) and rhp16(+) genes in Schizosaccharomyces pombe.

The global genome repair (GGR) subpathway of nucleotide excision repair (NER) is capable of removing lesions throughout the genome. In Saccharomyces cerevisiae the RAD7 and RAD16 genes are essential for GGR. Here we identify rhp7 (+), the RAD7 homolog in Schizosaccharomyces pombe. Surprisingly, rhp7 (+)and the previously cloned rhp16 (+)are located very close together and are transcribed in opposite directions. Upon UV irradiation both genes are induced, reaching a maximum level after 45-60 min. These observations suggest that the genes are co-regulated. Schizo-saccharomyces pombe rhp7 or rhp16 deficient cells are, in contrast to S.cerevisiae rad7 and rad16 mutants, not sensitive to UV irradiation. In S.pombe an alternative repair mechanism, UV damage repair (UVDR), is capable of efficiently removing photolesions from DNA. In the absence of this UVDR pathway both rhp7 and rhp16 deficient cells display an enhanced UV sensitivity. Epistatic analyses show that rhp7 (+)and rhp16 (+)are only involved in NER. Repair analyses at nucleotide resolution demonstrate that both Rhp7 and Rhp16, probably acting in a complex, are essential for GGR in S.pombe.     

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.     

The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions

Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protein (UV-DDB) has also been proposed as a damage recognition factor involved in repair of UV-photoproducts, especially CPD. Here, we show in human XP-E cells (UV-DDB deficient) that the incision complex formation at UV-induced lesions was severely diminished in locally damaged nuclear spots. Repair kinetics of CPD and 6-4PP in locally and globally UV-irradiated normal human and XP-E cells demonstrate that UV-DDB can mediate efficient targeting of XPC-hHR23B and other NER factors to 6-4PP. The data is consistent with a mechanism in which UV-DDB forms a stable complex when bound to a 6-4PP, allowing subsequent repair proteins--starting with XPC-hHR23B--to accumulate, and verify the lesion, resulting in efficient 6-4PP repair. These findings suggest that (i) UV-DDB accelerates repair of 6-4PP, and at later time points also CPD, (ii) the fraction of 6-4PP that can be bound by UV-DDB is limited due to its low cellular quantity and fast UV dependent degradation, and (iii) in the absence of UV-DDB a slow XPC-hHR23B dependent pathway is capable to repair 6-4PP, and to some extent also CPD.