Homologous and Nonhomologous Recombination Resulting in Deletion: Effects of p53 Status, Microhomology, and Repetitive DNA Length and Orientation

Abstract

Repetitive DNA elements frequently are precursors to chromosomal deletions in prokaryotes and lower eukaryotes. However, little is known about the relationship between repeated sequences and deletion formation in mammalian cells. We have created a novel integrated plasmid-based recombination assay to investigate repeated sequence instability in human cells. In a control cell line, the presence of direct or inverted repeats did not appreciably influence the very low deletion frequencies (2 × 10⁻⁷ to 9 × 10⁻⁷) in the region containing the repeat. Similar to what has been observed in lower eukaryotes, the majority of deletions resulted from the loss of the largest direct repeat present in the system along with the intervening sequence. Interestingly, in closely related cell lines that possess a mutant p53 gene, deletion frequencies in the control and direct-repeat plasmids were 40 to 300 times higher than in their wild-type counterparts. However, mutant p53 cells did not preferentially utilize the largest available homology in the formation of the deletion. Surprisingly, inverted repeats were approximately 10,000 times more unstable in all mutant p53 cells than in wild-type cells. Finally, several deletion junctions were marked by the addition of novel bases that were homologous to one of the preexisting DNA ends. Contrary to our expectations, only 6% of deletions in all cell lines could be classified as arising from nonhomologous recombination.

ACKNOWLEDGMENTS

We thank L. Reid for the kind gift of PNI2EX2, and we thank J. Haber and J. Nickoloff for helpful advice during the course of these experiments. Thanks are also due to J. B. Little, J. Carbon, and K. K. Hancock.

This work was supported by NIH grant CA49696. D.G. was supported by NIH training grant CA09078.