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Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB
V Rapić Otrin1, I Kuraoka, T Nardo
1Section on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-2725, USA.
Abstract:
Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.
Insights
Xeroderma pigmentosum group E (XP-E) cells show defects in UV-damaged DNA binding protein (UV-DDB) activity. Microinjection of UV-DDB partially corrected DNA repair in XP-E cells, suggesting its role in chromatin DNA repair.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- Xeroderma pigmentosum (XP) is a sun-sensitive disorder caused by defects in DNA repair.
- XP complementation groups A-G involve nucleotide excision repair (NER) pathway defects.
- XP group E (XP-E) exhibits mild symptoms with ~50% of normal DNA repair capacity.
Purpose of the Study:
- Investigate the roles of UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA) in XP-E.
- Clarify the specific molecular defect in newly identified XP-E cell lines.
- Determine the contribution of UV-DDB and RPA to DNA repair in XP-E.
Main Methods:
- Analyzed UV-DDB binding activity and RPA levels in XP-E cell lines.
- Assessed in vitro NER activity using UV-irradiated DNA and cisplatin lesions.
- Performed microinjection of purified UV-DDB into XP-E cells.
- Evaluated RPA's effect on DNA repair in various XP cell extracts.
Main Results:
- Newly identified XP-E cell lines showed reduced UV-DDB binding but normal RPA levels.
- XP-E cell extracts exhibited no significant in vitro NER defect with naked DNA.
- Microinjection of UV-DDB partially restored DNA repair in XP-E cells.
- RPA enhanced repair in normal and XP cells, indicating a non-specific effect.
Conclusions:
- The findings strengthen the link between XP-E and UV-DDB function.
- UV-DDB appears to play a crucial role in DNA repair within chromatin.
- This study elucidates a specific molecular defect contributing to the XP-E phenotype.