Widespread and nonrandom distribution of DNA palindromes in cancer cells provides a structural platform for subsequent gene amplification Hisashi Tanaka 1 , Donald A Bergstrom 2 , Meng-Chao Yao 1,3 & Stephen J Tapscott 4 Breakage-fusion-bridge cycles contribute to chromosome instability and generate large DNA palindromes that facilitate gene amplification in human cancers. The prevalence of large DNA palindromes in cancer is not known. Here, by using a new microarray-based approach called genome-wide analysis of palindrome formation, we show that palindromes occur frequently and are widespread in human cancers. Individual tumors seem to have a nonrandom distribution of palindromes in their genomes, and a subset of palindromic loci is associated with gene amplification. This indicates that the location of palindromes in the cancer genome can serve as a structural platform that supports subsequent gene amplification. Genome-wide analysis of palindrome formation is a new approach to identify structural chromosome aberrations associated with cancer. Amplification of large genomic regions is common in human cancers and contributes to tumor progression 1–3 . The incidence and distribu- tion of amplified loci has been well-documented in many tumors, but little is known about the mechanisms that determine whether a particular locus is susceptible to amplification. The initial formation of a large inverted repeat, or palindrome, is a rate-limiting step in gene amplification, and the molecular mechanisms that mediate palin- drome formation are conserved among eukaryotes 4–6 . Because break- age-fusion-bridge (BFB) cycles cause gene amplification in model systems 7–10 and palindrome formation is a structural component of BFB cycles, palindrome formation might mark regions susceptible to subsequent gene amplification. Using a new microarray-based approach for genome-wide analysis of palindrome formation (GAPF), we found that palindromes occur frequently in human cancers and seem to cluster at specific loci in the genome. Most palindrome-containing regions are not associated with gene amplification, but they are more likely to be amplified than are random loci. These data indicate that the formation of palindromes broadly alters the cancer genome and provides a structural platform for subsequent gene amplification. RESULTS GAPF We developed a new method to obtain a genome-wide assessment of palindrome formation based on the efficient intrastrand base pairing in large palindromic sequences 11,12 . Palindromic sequences can rapidly anneal intramolecularly to form ‘snap-back’ DNA under conditions that do not favor intermolecular annealing. We demonstrated this property by heat denaturation and rapid cooling of genomic DNA from D79IR-8 Sce2 cells containing two or three copies of a large DNA palindrome of the transgene DHFR (Fig. 1a) 4 . Southern-blot analysis showed that, in the presence of NaCl, renaturation occurred through intrastrand base-pairing, because the restriction fragments migrated at one-half the predicted size: the 10-kb KpnI fragment became 5 kb and the 22-kb XbaI fragment became 11 kb. To determine whether this snap-back property could be used to enrich for large palindromic sequences in total genomic DNA, we digested genomic DNA from D79IR-8 Sce2 cells with SalI, denaturated it, then rapidly renatured it with 100 mM NaCl and then digested it with the single-strand–specific nuclease S1 (Fig. 1b). Snap-back DNA formed from palindromes is double-stranded and should be resistant to S1, whereas the remainder of the genomic DNA is single stranded and should be sensitive to S1. We digested S1-resistant double- stranded DNA with MspI or TaqI, amplified it by ligation-mediated PCR (LM-PCR) using linker-specific primers and analyzed it by Sou- thern blotting with either a probe in the inverted repeat (probe 1) or a probe in an adjacent nonpalindromic fragment (probe 2). We detected a signal only with probe 1 in snap-back DNA, indicating that this method enriches palindromic sequences. We carried out a dilution experiment to test the ability of this technique to identify palindromes present in only a subpopulation of cells, as might occur in a tumor with a heterogeneous population of Published online 6 February 2005; doi:10.1038/ng1515 1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. 2 Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA. 3 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. 4 Division of Human Biology, Fred Hutchinson Cancer Research Center, Mailstop C3-168, 1100 Fairview Avenue North, Seattle, Washington 98109-1024, USA. Correspondence should be addressed to S.J.T. (stapscot@fhcrc.org) or M.-C.Y. (mcyao@imb.sinica.edu.tw). 320 VOLUME 37 [ NUMBER 3 [ MARCH 2005 NATURE GENETICS TECHNICAL REPORTS © 2005 Nature Publishing Group http://www.nature.com/naturegenetics