Nucleic Acids Research, 1990, Vol. 18, No. 20 5981-5988
© 1990
MOLECULAR BIOLOGY |
Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation)
1The Walther Oncology Center, Indiana University School of Medicine Indianapolis, IN 46202-5121, USA 2Departments of Pediatrics, Physiology and Biophysics, Indiana University School of Medicine Indianapolis, IN 46202-5121, USA 3Biochemistry and Molecular Biology, Indiana University School of Medicine Indianapolis, IN 46202-5121, USA
*To whom correspondence should be addressed
Received July 20, 1990. Revised September 23, 1990. Accepted September 23, 1990.
We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30 [EC] ), on illegitimate and extrachromosomal homologous recombination in mouse Ltk– cells. Cells were transfected with a wild type Herpes thymidine klnase (tk) gene or with two defective tk gene sequences followed by selection for tk-positive colonies. Using a wild type tk gene, colony formation required uptake, integration, and expression of the tk gene. Using defective tk genes, colony formation had the additional requirement for homologous recombination to reconstruct a functional tk gene. The presence of non-cytotoxic levels of 3-MB during and after transfection reduced the number of colonies recovered with a wild type tk gene in a dose-dependent manner, with 2 mM 3-MB causing a 10 to 20-fold reduction. 3-MB reduced the number of colonies recovered with defective tk genes only to the same extent as in transfections with a wild type gene. Treatment with 3-methoxybenzoic acid, a non-inhibitory analog of 3-MB, did not reduce the recovery of colonies in any experiment. Similar results were obtained using linear or supercoiled molecules and when defective tk genes were transfected into cells on one or two different DNA molecules. By assaying for transient expression of the tk gene, we found that 3-MB did not Inhibit uptake or expression of the tk gene. We conclude that poly(ADP-ribosylation) plays a role in random integration (illegitimate recombination) of DNA but does not play an important role in extrachromosomal homologous recombination, demonstrating that these two recombination pathways in cultured mouse fibroblasts are biochemically distinct.
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