Characterization of homologous DNA recombination activity in normal and immortal mammalian cells

Bhaskar Thyagarajan, Monica McCormick-Graham, Daniel P. Romero, Colin Campbell

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27 Scopus citations

Abstract

Homologous DNA recombination levels were measured in normal and spontaneously immortalized murine and human fibroblasts, and in a number of primate and murine established fibroblast cell lines. Immortal cell lines and tumor-derived clones homologously recombined extrachromosomal plasmid substrates at frequencies approximately 100-fold higher than did normal cells. To further explore the mechanism responsible for this phenotype, homologous recombination frequency was measured using nuclear extracts derived from normal and immortalized murine and human fibroblasts. Extracts prepared from immortal cells catalyzed high levels of homologous recombination, whereas very little recombination activity was detected in extracts prepared from normal fibroblasts. Similarly, only extracts derived from immortal cells contained strand-transferase activity as measured by the recently described pairing-on-membrane assay. Mixing experiments indicated that a recombination enhancing factor or factors present in immortal cells, rather than a recombination inhibitor in normal cells, was responsible for the enhanced homologous recombination activity observed using extracts derived from the former.

Original languageEnglish (US)
Pages (from-to)4084-4091
Number of pages8
JournalNucleic acids research
Volume24
Issue number20
DOIs
StatePublished - 1996

Bibliographical note

Funding Information:
This work was supported in part by grants from the Minnesota Medical Foundation, the H. Louise Ruddell Charitable Trust, the American Heart Association, MN affiliate, and the Leukemia Task Force of Minnesota. M. M.-G. was supported through funds provided by the University of Minnesota Graduate School. We are grateful to Dr Robert O’Dea for providing normal human fibroblasts, and for helpful discussions. Drs P.-Y. Law and L.-N. Wei provided immortalized monkey, and murine cell lines, respectively. We thank Drs Jennifer Cruise and Cecilia Warner for helpful editorial suggestions.

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