DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase.
Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.
Liu
J S
JS
Department of Microbiology & Biochemistry, State University of New York at Buffalo, School of Medicine & Biomedical Sciences, Buffalo, New York 14214, USA.
Kuo
S R
SR
Yin
X
X
Beerman
T A
TA
Melendy
T
T
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002461:Cell Line, Transformed / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects*; D004279:DNA, Viral / Q000037:antagonists & inhibitors / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053281:Enediynes; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051716:Replication Protein A
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D053281:Enediynes; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C058024:C 1027; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases