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Electroporation of normal human DNA endonucleases into xeroderma pigmentosum cells corrects their DNA repair defect
G J Tsongalis1, W C Lambert, M W Lambert
1Department of Pathology, UMDNJ, New Jersey Medical School, Newark.
Abstract:
Cells from patients with the cancer-prone inherited disease, xeroderma pigmentosum (XP) are known to be defective in the endonuclease-mediated incision step in excision repair of a number of different types of DNA adducts, but the molecular events responsible have not been delineated. We have previously reported isolation of two DNA endonucleases, pI 4.6 and 7.6, from normal human chromatin which recognize adducts produced by psoralen plus long wavelength ultraviolet radiation (UVA). These endonucleases are both present in XP complementation group A (XPA) cells even though these cells are hypersensitive to this type of damage. We now report that introduction by electroporation of either normal endonuclease into XPA cells restored their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS) to higher than normal levels following exposure to psoralen plus UVA. Introduction of XPA endonucleases into similarly treated XPA cells had little or no restorative effect on UDS. However, both normal and XPA endonucleases increased UDS in normal cells to higher than normal levels. These results indicate that XPA cells have endonucleases which can repair these adducts but which cannot function in intact cells unless a factor(s), which they lack is provided by normal cells.
Insights
Xeroderma pigmentosum (XP) cells have defective DNA repair. Introducing normal endonucleases into XP cells restored DNA repair, suggesting a missing factor in XP cells is required for endonuclease function.
Area of Science:
- Molecular biology
- Genetics
- Biochemistry
Background:
- Xeroderma pigmentosum (XP) is an inherited disease characterized by cancer predisposition.
- XP cells exhibit defects in DNA excision repair, particularly the endonuclease-mediated incision step.
- Specific DNA endonucleases recognizing psoralen-UVA adducts were previously isolated from normal human cells.
Purpose of the Study:
- To investigate the molecular basis of DNA repair defects in XP complementation group A (XPA) cells.
- To determine the functional role of specific DNA endonucleases in the repair of psoralen-UVA induced DNA adducts.
- To identify potential factors missing in XPA cells that regulate endonuclease activity.
Main Methods:
- Electroporation of DNA endonucleases into XPA cells.
- Assessing unscheduled DNA synthesis (UDS) as a measure of DNA repair capacity.
- Comparing UDS levels in XPA cells and normal cells following psoralen-UVA exposure and endonuclease introduction.
Main Results:
- Introduction of normal endonucleases into XPA cells significantly restored UDS following psoralen-UVA treatment.
- XPA endonucleases showed little restorative effect on UDS in XPA cells.
- Both normal and XPA endonucleases enhanced UDS in normal cells, indicating a requirement for additional factors in XPA cells.
Conclusions:
- XPA cells possess functional endonucleases for psoralen-UVA adduct repair, but their activity is impaired in intact cells.
- A factor(s) present in normal cells, but lacking in XPA cells, is necessary for proper endonuclease function in DNA repair.
- This finding elucidates a critical aspect of the molecular mechanism underlying DNA repair deficiencies in Xeroderma Pigmentosum.