Single-Stranded Annealing Induced by Re-Initiation of Replication Origins Provides a Novel and Efficient Mechanism for Generating Copy Number Expansion via Non-Allelic Homologous Recombination

Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA) suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR). Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA) mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA–mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution.

拷贝数扩增（Copy number expansion），包括扩增与重复，可促成人类表型变异，推动进化过程中的分子多样化，并驱动多种癌症的起始及/或进展。然而，此类拷贝数改变背后的分子机制尚未完全阐明。我们近期证实，在酿酒酵母（Saccharomyces cerevisiae）单个复制起点处发生的短暂、受限的再复制过程，可高效诱导再复制区域的节段性扩增。针对此类再复制诱导的基因扩增（Re-replication Induced Gene Amplification, RRIGA）的结构分析显示，RRIGA可通过非等位同源重组（Non-allelic Homologous Recombination, NAHR）为拷贝数变异（Copy number Variation, CNV）的产生提供全新机制。本研究阐明了这一全新机制，并揭示了其极高效率的内在原因。我们证实，序列同源性是重复元件参与RRIGA的必要且充分条件，并证明二者的重组通过单链退火（Single-strand Annealing, SSA）机制完成。我们还发现，再复制叉（Re-replication Fork）易发生断裂，这可解释复制蛋白失调相关的广泛DNA损伤现象。此类断裂似乎可刺激再复制起始位点侧翼的再复制重复序列之间发生NAHR。我们的研究结果支持如下RRIGA模型：再复制泡（Re-replication Bubble）的扩增范围超出侧翼同源序列后，反式作用下的两个复制叉发生断裂，可为SSA介导的NAHR形成首尾相连的重复提供理想的结构环境。鉴于RRIGA具有极高的发生效率，我们认为其可能是疾病与进化过程中未被认知的拷贝数扩增诱因之一。