Mitotic recombination in mammalian cells

Abstract: Recombination is usually defined as the exchange of genetic material between two strands or regions of nucleic acids. This process occurs in all known organisms and is highly conserved, especially among higher eukaryotes. Various types of recombination, involving homologous or non-homologous nucleic acid sequences, are known to exist. Recombination is a double-edged sword that may be beneficial or harmful for the cell. On one hand, it fulfills essential functions in connection with, e.g., repair of DNA double- strand breaks and maintenance of genomic stability; but, at the same time, this process is also partly responsible for, among other things, error prone repair and genomic instability, which can lead to cancer.The aim of the present study has been to investigate molecular mechanisms underlying spontaneous and induced mitotic recombination in mammalian cells and, in particular, to characterize the role of the RAD51 protein in these processes. For this purpose, V79 Chinese hamster cell lines containing spontaneous partial duplications of the hprt gene were employed. A new approach to investigate homologous recombination, which offers the unique possibility of determining the type of homologous recombination involved, was developed. This assay procedure was compared to other systems used previously for detection of induced recombination. Use of this newly developed method to characterize mechanisms underlying induction of homologous recombination revealed that inhibition of DNA synthesis is a potent pathway for such induction.Subsequently, the effect of overexpressing RAD51 on two different assays for recombination was determined. Our findings suggest that the RAD51 protein supports spontaneous homologous recombination via an exchange mechanism, as well as being involved in spontaneous non-homologous recombination, possibly with respect to class switch recombination. However, RAD51 was found not to affect induced non-homologous recombination, suggesting that this protein might not be involved in repairing DNA damage via non-homologous end-joining.Finally, the repair of DNA double-strand breaks induced in the S phase of the cell cycle was examined. Our observations in this case suggest that homologous recombination by strand invasion, employing an exchange mechanism, is a major feature of such repair and, furthermore, that a functional pathway for recombination is essential for the survival of cells in which DNA double-strand breaks have occurred.In summary, the work described here improves our understanding of the molecular mechanisms underlying spontaneous and induced recombination, as well as the repair of DNA double-strand breaks in mammalian cells.