Related Experiment Videos
Radiation effects on DNA synthesis in a defined chromosomal replicon
J M Larner1, H Lee, J L Hamlin
1Department of Radiology, University of Virginia School of Medicine, Charlottesville 22908.
Molecular and Cellular Biology
|March 1, 1994
Summary
DNA damage triggers a p53-independent pathway that halts DNA replication initiation at specific origins within 30 minutes. This rapid response occurs independently of the known G1 cell cycle arrest.
Area of Science:
- Cellular Biology
- Molecular Biology
- Radiation Biology
Background:
- The tumor suppressor p53 mediates a DNA damage response, causing cell cycle arrest in late G1.
- This p53 pathway does not fully explain the rapid reduction in DNA synthesis post-irradiation.
Purpose of the Study:
- Investigate a p53-independent DNA damage response pathway affecting DNA synthesis.
- Characterize the acute radiation effect on DNA replication initiation at a specific locus.
Main Methods:
- Utilized the amplified dihydrofolate reductase (DHFR) domain in methotrexate-resistant CHO cells (CHOC 400) as a model replicon.
- Employed two-dimensional gel replicon mapping to study DNA initiation.
- Used the replication inhibitor mimosine to define critical cell cycle positions.
Main Results:
- CHOC 400 cells exhibit an acute-phase response but lack p53-mediated G1 arrest.
- Irradiation (900 cGy) completely inhibited DHFR locus initiation within 30 minutes, resuming after 3-4 hours.
- Identified a p53-independent pathway regulating initiation at individual origins during S-phase.
Conclusions:
- Radiation quantitatively down-regulates DNA initiation at a defined chromosomal origin.
- A p53-independent S-phase damage-sensing pathway operates at the level of individual replication origins.
- Cells cannot prevent initiation at early-firing origins post-G1/S boundary following irradiation.