School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.
PLoS Genet. 2012;8(12):e1003119. doi: 10.1371/journal.pgen.1003119. Epub 2012 Dec 13.
DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature.
DNA 扩增是一种分子过程，可增加染色体片段的拷贝数，并经常导致扩增基因的表达升高。尽管基因扩增在癌症和其他退行性疾病中经常观察到，但涉及 DNA 拷贝数增加的分子机制在很大程度上仍不清楚。我们假设小的 DNA 片段可能是 DNA 扩增事件的触发因素。在发现小的 DNA 片段（如 DNA 寡核苷酸）可以高度重组之后，我们在酵母酿酒酵母中开发了一种系统，以捕获由小 DNA 片段引发的染色体 DNA 扩增事件。在这里，我们证明小 DNA 可以扩增染色体区域，产生串联重复或无着丝粒的额外染色体 DNA 环。小片段驱动的 DNA 扩增（SFDA）的发生频率随小 DNA 与靶染色体区域之间同源性的长度而增加。即使小的单链分子与基因组靶标具有 20-nt 的同源性，SFDA 事件也会被触发。位于染色体扩增区域之外的双链断裂（DSB）可将扩增事件的频率提高 20 倍，并有利于形成额外的染色体环。SFDA 依赖于 Rad52 和 Rad59，部分依赖于 Rad1、Rad10 和 Pol32，独立于 Rad51，表明这是一种单链退火机制。我们的结果揭示了一种新的基因扩增分子模型，其中小 DNA 片段驱动 DNA 扩增并定义了扩增区域的边界。由于 DNA 片段经常在细胞内和细胞外环境中被发现，例如癌症或其他退行性疾病患者的血清中，我们提出 SFDA 可能是癌细胞中 DNA 扩增的常见机制，也是自然界中 DNA 拷贝数变异的更普遍原因。
