Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome.
Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC(50) of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases alpha, delta and epsilon is 15, 45 and 125 muM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC(50)=88 muM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.
Department of Medicine, Hematology/Oncology Division, Indiana University School of Medicine, Indiana University Cancer Research Institute, 1044 W. Walnut Street R4-170 Indianapolis, IN 46202, USA.
Heqiao
H
Dai
Jianying
J
Liu
Linda H
LH
Malkas
Jennifer
J
Catalano
Srilakshmi
S
Alagharu
Robert J
RJ
Hickey
D002453:Cell Cycle; D002857:Chromium / Q000633:toxicity*; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004785:Environmental Pollutants / Q000633:toxicity*; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000187:drug effects*; D013997:Time Factors; D000099272:Replisomes
D002857:Chromium; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004785:Environmental Pollutants; D009097:Multienzyme Complexes; D000099272:Replisomes