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Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).

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  • 1. Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202-5121.
      Authors
    • Waldman BC 1
    (1 author)

Nucleic Acids Research, 01 Oct 1990, 18(20):5981-5988
https://doi.org/10.1093/nar/18.20.5981 PMID: 2172923 PMCID: PMC332394

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Abstract 

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (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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Nucleic Acids Res. 1990 Oct 25; 18(20): 5981–5988.
PMCID: PMC332394
PMID: 2172923

Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).

B C Waldman and A S Waldman
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Abstract

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
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