genes G C A T T A C G G C A T Review Gene Amplification and the Extrachromosomal Circular DNA Noriaki Shimizu   Citation: Shimizu, N. Gene Amplification and the Extrachromosomal Circular DNA. Genes 2021, 12, 1533. https://doi.org/ 10.3390/genes12101533 Academic Editors: Eishi Noguchi and Maciej Wnuk Received: 7 August 2021 Accepted: 23 September 2021 Published: 28 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Graduate School of Integrated Sciences for Life, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Hiroshima, Japan; shimizu@hiroshima-u.ac.jp Abstract: Oncogene amplification is closely linked to the pathogenesis of a broad spectrum of human malignant tumors. The amplified genes localize either to the extrachromosomal circular DNA, which has been referred to as cytogenetically visible double minutes (DMs), or submicroscopic episome, or to the chromosomal homogeneously staining region (HSR). The extrachromosomal circle from a chromosome arm can initiate gene amplification, resulting in the formation of DMs or HSR, if it had a sequence element required for replication initiation (the replication initiation region/matrix attachment region; the IR/MAR), under a genetic background that permits gene amplification. In this article, the nature, intracellular behavior, generation, and contribution to cancer genome plasticity of such extrachromosomal circles are summarized and discussed by reviewing recent articles on these topics. Such studies are critical in the understanding and treating human cancer, and also for the production of recombinant proteins such as biopharmaceuticals by increasing the recombinant genes in the cells. Keywords: gene amplification; extrachromosomal DNA; double minutes; micronucleus; cancer; genome plasticity; chromothripsis; gene expression; repeat-induced gene silencing 1. Gene Amplification and the Extrachromosomal Circles in Human Cancer The amplification of oncogenes or drug-resistant genes plays a pivotal role in human cell malignant transformation by conferring growth advantage to the cells through the overproduction of the amplified gene product. A classical cytogenetic study located the amplified genes at the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR) [1]. DMs and HSR mutually interconvert [2,3], and share the same sequence [4]. DMs are stable extrachromosomal elements that contain circular DNA. Circularity has been suggested based on electron microscopy [5], sensitivity to radiation-mediated breakage [6], and the absence of telomeric structures [7]; this was recently re-enforced by integrating ultrastructural imaging, long-range optical mapping, and computational analysis of whole-genome sequencing [8]. In contrast, cytogenetically undetectable circular DNA has been identified in many normal and cancer cell lines and normal tissues more than three decades ago [9]. Recently, many reports have described circular extrachromosomal DNA in normal or cancer cells [10]. In general, the circles in normal cells [11,12] were smaller in size (less than 1 kbp) than those in cancer cells (1–2 Mbp) [13]. The former is referred to as extrachromosomal closed circular DNA (eccDNA), and the latter are referred to as extrachromosomal DNA (ecDNA). EcDNAs are equivalent to conventional DMs; however, the term ecDNA was recently used instead of DMs because it does not always appear as a doublet among the chromosome spread specimens. Several extensive studies that used a large number of clinical samples together with the most advanced techniques, unambiguously, reinforced the tight relationship between malignancy and the appearance of ecDNA/DMs [13,14]. It is important to note that gene expression from the same amplicon sequence is higher in the extrachromosomal context than in the chromosomal context [15] because the chromatin of extrachromosomal DNA is more favorable for gene expression [8,16]. Consistently, DMs were replicated early in the S phase, while the HSRs of the same Genes 2021, 12, 1533. https://doi.org/10.3390/genes12101533 https://www.mdpi.com/journal/genes