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The Cell Cycle pp 167-171 | Cite as

Preferential Repair of Cisplatin Adducts in the Human DHFR Gene During G1 Phase Assayed with T4 DNA Polymerase

  • Nicholas J. Rampino
  • Vilhelm A. Bohr
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)

Summary

We use a novel assay to detect strand specific repair in genes after cellular exposure to cisplatin at IC50 levels. Single stranded DNA capable of hybridizing to gene specific probes is generated enzymatically by the 3′–5′ exonuclease activity of 74 DNA polymerise. in the presence of cisplatin adducts, the exonuclease activity of this enzyme is blocked, preventing the formation of single stranded DNA, and thus lowering the amount of complementary sequence available for probe hybridization.1,2

Human ovarian carcinoma cells synchronized in M phase were treated at the beginning of G1 phase with 5uM cisplatin for 1 hr, and then allowed to repair in drug free media. Repair during early G1 phase in the unexpressed δ-globin gene was compared to that in a late growth-regulated gene, dihydrofolate reductase, that is involved in nucleotide biosynthesis, and normally shows induction at the G1 -S boundary. Extensive cisplatin adduct removal was measured in the dihydrofolate reductase gene after four hours of cellular repair, whereas there was no evidence of repair in the δ-globin gene.

Finally, we show that this assay utilizing T4 DNA polymerase can detect low levels of DNA oxidation, brought about by cellular exposure to hydrogen peroxide.

Keywords

Proliferate Cell Nuclear Antigen Exonuclease Activity Drug Free Medium Human Ovarian Carcinoma Cell DHFR Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1994

Authors and Affiliations

  • Nicholas J. Rampino
    • 1
  • Vilhelm A. Bohr
    • 1
  1. 1.Laboratory of Molecular GeneticsNIA, NIHBaltimoreUSA

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