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Glycosylases that excise modified DNA pyrimidines in young and senescent human WI-38 fibroblasts
1Department of Pathology, Temple University School of Medicine, Philadelphia, PA 19140.
Abstract:
Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair in human cells is initiated by DNA glycosylases which remove oxidized bases from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme has glycosylic activity against many ring-saturated DNA pyrimidines. Levels of these enzymes were examined in WI-38 fibroblasts of different culture ages. All glycosylases were assayed by measurements of direct release of modified free bases from their respective DNA substrates. Levels of 5-hydroxymethyluracil-DNA glycosylase were reduced in aging cells. Specific activities of the glycosylase that releases ring-saturated pyrimidines and of uracil-DNA glycosylase were not substantially altered in senescent cells. Therefore, although aging cells might have reduced excision of DNA 5-hydroxymethyluracil, there is no overall age-dependent decrease of DNA glycosylase activities.
Insights
Aging cells show reduced 5-hydroxymethyluracil-DNA glycosylase activity, impacting DNA repair. However, overall DNA glycosylase activity remains stable in senescent cells, suggesting no general decline in DNA repair enzymes.
Area of Science:
- Molecular Biology
- Cellular Aging
- DNA Repair Mechanisms
Background:
- Cellular DNA integrity is crucial and constantly challenged by oxidative damage.
- DNA glycosylases initiate base excision repair by removing damaged bases.
- Key enzymes include 5-hydroxymethyluracil-DNA glycosylase and others acting on ring-saturated pyrimidines.
Purpose of the Study:
- To investigate age-dependent changes in DNA glycosylase activities in human fibroblasts.
- To determine if specific DNA glycosylases decline with cellular senescence.
- To assess the overall impact of aging on base excision repair initiation.
Main Methods:
- Assaying DNA glycosylase activity by measuring the direct release of modified free bases from DNA substrates.
- Comparing enzyme levels in WI-38 fibroblasts at different culture ages (young vs. senescent).
- Quantifying the activity of 5-hydroxymethyluracil-DNA glycosylase, uracil-DNA glycosylase, and a glycosylase for ring-saturated pyrimidines.
Main Results:
- Levels of 5-hydroxymethyluracil-DNA glycosylase were found to be reduced in aging WI-38 fibroblasts.
- Specific activities of uracil-DNA glycosylase and the glycosylase for ring-saturated pyrimidines showed no significant alteration in senescent cells.
- These findings indicate a specific age-related decrease in one type of DNA glycosylase activity.
Conclusions:
- Aging human cells exhibit a diminished capacity for excising 5-hydroxymethyluracil from DNA due to reduced enzyme levels.
- Despite the decrease in 5-hydroxymethyluracil-DNA glycosylase, other DNA glycosylase activities remain stable.
- There is no overall age-dependent decline in DNA glycosylase activities, suggesting compensatory mechanisms or specific vulnerabilities in DNA repair during aging.