De novo‐generated small palindromes are characteristic of amplicon boundary junction of double minutes

Abstract

Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.

Ribosomal L22-like1 (RPL22L1) Promotes Ovarian Cancer Metastasis by Inducing Epithelial-to-Mesenchymal Transition

“…Cells were harvested for metaphase spread preparation according to the methods described in previous studies [ 14 , 15 ] and were stained with Giemsa. Two BAC clones, GFP-RP11-355H10, which specifically covers the MYCN (amplified in UACC-1598 and located on the DMs [ 13 ]), and Cy3-RP11-726H11 for RPL22L1 , were selected as DNA probes and hybridized to metaphase spreads of cells as described previously [ 16 ]. Chromosomes were counterstained with DAPI (4, 6-diamidino-2-phenylindole).…”

“…Our team previously identified 3q26.2 as an origin of DMs in the human ovarian cancer cell line UACC-1598, and a series of genes were co-amplified on the same ovarian DMs, including MYCN , EIF5A2 , and RPL22L1 [ 13 ]. Both MYCN and EIF5A2 play important roles in cancer progression.…”

Cell-Free eccDNAs: A New Type of Nucleic Acid Component for Liquid Biopsy?

“…This entire procedure is illustrated in Figure 1. Although this strategy is very effective for small eccDNAs (like microDNA and spcDNA) it remains difficult to extract the entire circular DNA for the large DMs, for which alternative methods like microdissection (13), array-CGH (39) or direct sequencing of the total DNA (40) can be applied, as sequences on DMs are highly amplified. As the quantity of cfDNAs obtained from body fluids is very limited, when using cfDNAs as starting material, eccDNA enrichment method may need further improvement to increase sensitivity.…”

“…To unify the nomenclature, Moller et al has proposed to use extrachromosomal circular DNAs (eccDNAs) to describe all circular DNAs in eukaryotes (6). The eccDNAs have different sizes, including large circular DNAs such as episomes (10, 11) and double minute chromosomes (DMs) (12, 13), and the smaller circular molecules such as the small polydispersed DNA (spcDNAs) (14, 15) and microDNAs (5, 16). Episomes (from hundreds of kilobases to 1 megabase, submicroscopic) are considered as precursors of DMs (>1 megabase, microscopically visible).…”

“…In pathological conditions, eccDNAs are found to play roles in human diseases and disease treatments, especially for larger eccDNAs (episomes and DMs) with their functions being better characterized. For example, DMs, exclusively found in tumor cells (13) and drug-resistance cells (43), significantly elevate the active gene transcripts to promote tumor progression. Because of their self-replication and uneven segregation, DMs are related to tumor cell heterogeneity and adaptation (44).…”