Chromosome Breakage and Large DNA Palindrome Formation in Mammalian Cells
The specific aim of this project between the Yao lab and the Tapscott lab is to understand the molecular basis of gene amplification in mammalian cells. Amplified genes can often reside extrachromosomally in double minute chromosomes as large inverted repeats (palindromes). Although the occurrences of this process have been well documented, its molecular mechanism remains unsolved. Studies of the Yao lab in Tetrahymena and yeast have revealed that large DNA palindrome formation requires a DNA double strand break (DSB) at the end of the amplicon and a pair of short inverted repeats (SIR) next to the break. In this collaboration, we have been testing if DSB and SIR play a critical role for palindrome formation in mammalian cells as they do in Tetrahymena. Dehydrofolate reductase (DHFR)-deficient Chinese Hamster Ovary cells were transformed with a vector containing an I-SceI cutting site, 77 or 229 base-pairs of SIR, and the mouse DHFR gene. We found the large palindromic DHFR transgenes after DSB whose junction was exactly the same as we have predicted. In contrast, we didn't find any palindrome from noIRSce transformants, suggesting the possible role of SIR for the palindrome formation in mammalian cells.
To confirm the important role of SIR, we will create the transformants with or without SIR in the same genomic locus by the Cre-loxP system. We will also try to see palindrome formation right after DSB by PCR-based physical analysis. By using FISH and pulse field gel electrophoresis, we will characterize these amplicons. We are also working on another construct that contains (in the following order) a PGK promoter-splice donor, an inverted splice acceptor-Beta-geo-pA cassette, 77bp SIR, an I-SceI cutting site and a CMV HyTk cassette. This construct will be used in a variety of cell lines with different genetic baqckgrounds to study the involvement of those genes in palindromic formation. To further test our idea, we will determine the nucleotide sequence at the junction of the amplification unit in a naturally occurring palindrome, such as the c-myc amplicon in the HL60 cell line.