Related Experiment Videos
Decreased rates of replicon initiation in mammalian cells
1Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Abstract:
We have designed an assay to measure the rate of initiation of DNA synthesis in Friend erythroleukemia cells and have shown that this parameter is reduced by gamma-radiation and treatment with 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylene-beta-D-glucopyranoside) (VP-16). It is concluded, that double-strand breaks in DNA are the immediate cause for this effect. The decrease in the rate of replicon initiation is affected differently by different agents such as cis-diamminedichloroplatinum(II), cycloheximide, staurosporine, and 3-aminobenzamide. The analysis of these results indicates that the observed partial decrease of the rate of DNA initiation is most probably transmitted from the site of damage to the initiation site by one or more phosphorylation/dephosphorylation steps. It does not require de novo synthesis of protein factors, but is probably dependent on poly(ADP-ribosyl)ation of chromatin at the site of DNA breaks.
Insights
DNA double-strand breaks, induced by radiation and VP-16, reduce DNA synthesis initiation in erythroleukemia cells. This effect is mediated by phosphorylation and poly(ADP-ribosyl)ation, not new protein synthesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Radiation Biology
Background:
- DNA synthesis initiation is crucial for cell proliferation.
- DNA damage can impede cellular processes.
- Friend erythroleukemia cells provide a model for studying DNA synthesis.
Purpose of the Study:
- To develop an assay for measuring DNA synthesis initiation rate.
- To investigate the impact of DNA-damaging agents on DNA synthesis.
- To elucidate the molecular mechanisms underlying the reduction in DNA synthesis initiation.
Main Methods:
- Developed a novel assay to quantify DNA synthesis initiation rate.
- Treated Friend erythroleukemia cells with gamma-radiation and VP-16.
- Administered various DNA-damaging agents including cis-diamminedichloroplatinum(II), cycloheximide, staurosporine, and 3-aminobenzamide.
Main Results:
- Gamma-radiation and VP-16 significantly reduced DNA synthesis initiation rate.
- DNA double-strand breaks were identified as the direct cause of this reduction.
- Different agents affected the rate of replicon initiation distinctively.
Conclusions:
- The reduction in DNA synthesis initiation is linked to DNA double-strand breaks.
- Signal transduction likely involves phosphorylation/dephosphorylation events.
- Poly(ADP-ribosyl)ation at DNA break sites, rather than de novo protein synthesis, appears critical.