Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells

Abstract

Gene amplification is a common form of genomic instability in a wide variety of organisms and is often associated with tumor progression in mammals. One striking feature of many amplified genes is their organization as large inverted duplications (palindromes). Here, we describe a molecular mechanism for palindrome formation in mammalian cells that is also conserved in protists. We introduced a short (79 or 229 bp) inverted repeat into the genome of Chinese hamster ovary cells and showed that it promoted the formation of a large DNA palindrome after an adjacent DNA double-strand break. This finding suggests that short inverted repeats in the mammalian genome can have a critical role in the initiation of gene amplification. This specific mechanism may provide a novel target for cancer therapies.

Genetic determinants of methotrexate responsiveness and resistance in colon cancer cells

“…A 20-40-fold amplification of the DHFR gene was demonstrated by real-time PCR (see Supplementary Information). DNA amplification has been associated with chromosome double-strand breaks or activation of fragile sites, and these chromosomal abnormalities are caused by structural chromosomal instability in human tumors (Yunis and Soreng, 1984;Poupon et al, 1996;Coquelle et al, 1997;Singer et al, 2000;Tsushimi et al, 2001;Tanaka et al, 2002). HT-29, SW480 and SK-CO-1 cells fitted within this hypothesis because all of them displayed aneuploid complex karyotypes and exhibited pre-existing amplifications.…”

Intrastrand Annealing Leads to the Formation of a Large DNA Palindrome and Determines the Boundaries of Genomic Amplification in Human Cancer

“…To construct pD229IRScelox2, primer pairs, either XbaIIR and XholoxIR or XhoCenPt and PstloxIR, were used to amplify each one of the two parts of the 229-base-pair inverted repeat (229IR) with pD229IRSce as a template (37). PCR products were cloned into pCRII-TOPO (Invitrogen) using the TOPO TA cloning kit to generate pTOPOXbaIR and pTOPOPstIR.…”

“…In a prior study, we showed that a short DNA IR adjacent to a DSB could catalyze the formation of a large palindrome and subsequent gene amplification in mammalian cells (37). Thus, gene amplification in cancer cells might proceed through the same intramolecular pathway as the developmentally regulated gene amplification in single-cell eukaryotes.…”

“…In fact, either a chromosome break at a fragile site or a site-specific chromosomal DNA double-strand break (DSB) induced by I-SceI endonuclease leads to gene amplification by initiating the formation of large palindromic chromosomes (9,37). Large palindromic dicentric chromosomes generate further chromosome breaks during chromosome segregation (see Fig.…”

High-Resolution ROMA CGH and FISH Analysis of Aneuploid and Diploid Breast Tumors

“…Clearly, the process requires some structural characteristic of the recombining chromosome arms. Whether the key to that process resides in telomere loss or in recombination at short inverted repeats (Tanaka et al 2002) or through a related mechanism, break-induced replication, where segments are copied from internal chromosome breaks (Difilippantonio et al 2002), is as yet an open question. It will be most interesting to determine whether a component of that peculiar cancer-related process can be blocked, thus providing another target for anticancer therapy.…”