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Replication of damaged DNA in vitro is blocked by p53
1Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Abstract:
The tumor suppressor protein p53 may have other roles and functions in addition to its well-documented ability to serve as a sequence-specific transcriptional activator in response to DNA damage. We showed previously that p53 can block the replication of polyomavirus origin-containing DNA (Py ori-DNA) in vitro when p53 binding sites are present on the late side of the Py ori. Here we have both further extended these observations and have also examined whether p53 might be able to bind directly to and inhibit the replication of damaged DNA. We found that p53 strongly inhibits replication of gamma-irradiated Py ori-DNA and such inhibition requires both the central DNA binding domain and the extreme C-terminus of the p53 protein. An endogenous p53 binding site lies within the Py origin and is required for the ability of p53 to block initiation of replication from gamma-irradiated Py ori-DNA, suggesting the possibility of DNA looping caused by p53 binding both non-specifically to sites of DNA damage and specifically to the endogenous site in the polyomavirus origin. Our results thus suggest the possibility that under some circumstances p53 might serve as a direct regulator of DNA replication and suggest as well an additional function for cooperation between its two autonomous DNA binding domains.
Insights
The tumor suppressor protein p53 inhibits DNA replication of damaged polyomavirus DNA. This function requires specific domains of p53 and may involve DNA looping for regulation.
Area of Science:
- Molecular Biology
- Virology
- Cancer Research
Background:
- The tumor suppressor protein p53 is known for its role as a transcriptional activator following DNA damage.
- Previous studies indicated p53 can inhibit polyomavirus origin DNA replication (Py ori-DNA) in vitro when binding sites are present.
Purpose of the Study:
- To investigate if p53 can directly bind to and inhibit the replication of damaged DNA.
- To further extend previous observations on p53's inhibitory role in DNA replication.
Main Methods:
- In vitro replication assays using gamma-irradiated polyomavirus origin-containing DNA (Py ori-DNA).
- Analysis of p53 protein domains required for inhibition, focusing on the central DNA binding domain and C-terminus.
- Examination of p53 binding to an endogenous site within the polyomavirus origin.
Main Results:
- p53 strongly inhibits the replication of gamma-irradiated Py ori-DNA.
- Inhibition requires both the central DNA binding domain and the C-terminus of p53.
- An endogenous p53 binding site within the Py origin is crucial for blocking replication initiation from damaged DNA.
Conclusions:
- p53 may act as a direct regulator of DNA replication, particularly for damaged DNA.
- The findings suggest a potential mechanism involving DNA looping, where p53 binds to both damaged DNA sites and specific origin sequences.
- This highlights a cooperative function between p53's DNA binding domains in regulating DNA replication.