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Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision
J P Therrien1, R Drouin, C Baril
1Division of Pathology, Department of Medical Biology, Faculty of Medicine, Laval University, Quebec, Canada.
Abstract:
After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li-Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G(1) arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li-Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-jun loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair.
Insights
The tumor suppressor p53 is crucial for DNA repair, including transcription-coupled repair, after UV exposure. Its absence or inactivation by HPV E6 impairs DNA repair, while HPV E7 affects global repair.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The p53 tumor suppressor is vital in preventing cancer by controlling cell cycle arrest and apoptosis after DNA damage.
- Previous studies indicated p53's role in global nucleotide excision repair (NER) but not transcription-coupled NER (TC-NER) in UV-exposed cells.
- The human papillomavirus (HPV) E6 and E7 oncoproteins are known to inactivate p53 and pRb, respectively, contributing to carcinogenesis.
Purpose of the Study:
- To investigate the role of p53 in both global NER and TC-NER following UV radiation.
- To examine the impact of HPV E6 and E7 oncoproteins on DNA repair pathways in human fibroblasts.
- To elucidate the DNA repair mechanisms affected by p53 inactivation and its relevance in HPV-mediated cancers.
Main Methods:
- Utilized ligation-mediated PCR for high-resolution quantification of UVB-induced cyclobutane pyrimidine dimer (CPD) repair.
- Assessed DNA repair in p53-deficient Li-Fraumeni skin fibroblasts.
- Analyzed DNA repair in human lung fibroblasts expressing HPV E6 (p53 inactivation) or HPV E7 (pRb inactivation).
Main Results:
- p53-deficient cells (Li-Fraumeni) and HPV E6-expressing cells showed defective CPD repair in both transcribed and nontranscribed strands at specific gene loci.
- HPV E7-expressing cells exhibited reduced CPD removal exclusively in the nontranscribed strand.
- These findings indicate that both global NER and TC-NER are dependent on p53 function in UV-exposed human fibroblasts.
Conclusions:
- Transcription-coupled repair is a p53-dependent process in UV-exposed human fibroblasts, challenging previous assumptions.
- The DNA repair defect observed in HPV E7-expressing cells suggests a role for this oncoprotein in HPV-related carcinogenesis.
- The study implies a potential involvement of the retinoblastoma tumor-suppressor protein (pRb) in global nucleotide excision repair.