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The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway
X W Wang1, W Vermeulen, J D Coursen
1Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Abstract:
The molecular pathway of p53-dependent apoptosis (programmed cell death) is poorly understood. Because p53 binds to the basal transcription-repair complex TFIIH and modulates its DNA helicase activities, we hypothesized that TFIIH DNA helicases XPB and XPD are members of the p53-mediated apoptotic pathway. Whereas transfer of a wild-type p53 expression vector by microinjection or retroviral infection into primary normal human fibroblasts resulted in apoptosis, primary fibroblasts from individuals with xeroderma pigmentosum (XP), who are deficient in DNA repair and have germ-line mutations in the XPB or XPD gene, but not in the XPA or XPC gene, have a deficiency in the apoptotic response. This deficiency can be rescued by transferring the wild-type XPB or XPD gene into the corresponding mutant cells. XP-D lymphocytes also have a decreased apoptotic response to DNA damage by adriamycin, indicating a physiologically relevant deficiency. The XP-B or XP-D mutant cells undergo a normal apoptotic response when microinjected with the Ich-L, and ICE genes. Analyses of p53 mutants and the effects of microinjected anti-p53 antibody, Pab421, indicate that the carboxyl terminus of p53 may be required for apoptosis. Direct microinjection of the p53 carboxy-terminal-derived peptide (amino acid residues 319-393) resulted in apoptosis of primary normal human fibroblasts. These results disclose a novel pathway of p53-induced apoptosis.
Insights
This study reveals a new pathway for programmed cell death (apoptosis) involving the p53 protein. It identifies DNA repair proteins XPB and XPD as key players in p53-mediated apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The molecular mechanisms of p53-dependent apoptosis are not fully understood.
- The p53 protein interacts with the transcription-repair complex TFIIH, influencing its DNA helicase activity.
Purpose of the Study:
- To investigate the role of TFIIH DNA helicases XPB and XPD in the p53-mediated apoptotic pathway.
- To elucidate the specific domains of p53 involved in inducing apoptosis.
Main Methods:
- Microinjection and retroviral infection of wild-type p53 into human fibroblasts.
- Analysis of apoptosis in fibroblasts from xeroderma pigmentosum (XP) patients with mutations in XPB or XPD.
- Gene rescue experiments by transferring wild-type XPB or XPD genes.
- Apoptosis assays using adriamycin and microinjection of specific p53 peptides or antibodies.
Main Results:
- Primary fibroblasts deficient in XPB or XPD showed impaired p53-induced apoptosis, unlike those deficient in XPA or XPC.
- The apoptotic deficiency in XP-B and XP-D cells could be rescued by introducing the corresponding wild-type genes.
- XP-D lymphocytes exhibited reduced apoptosis following adriamycin treatment.
- Microinjection of a peptide derived from the p53 carboxyl terminus induced apoptosis in normal fibroblasts.
Conclusions:
- TFIIH DNA helicases XPB and XPD are integral components of the p53-dependent apoptotic pathway.
- The carboxyl terminus of p53 is crucial for mediating apoptosis.
- This research uncovers a novel molecular pathway for p53-induced programmed cell death.