Single-strand annealing between inverted DNA repeats: Pathway choice, participating proteins, and genome destabilizing consequences

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

双链DNA断裂（double strand DNA breaks, DSBs）是一类危险的基因组事件，可由多种因素诱发，包括环境诱变损伤或DNA复制叉崩溃。尽管高效且精准的双链DNA断裂修复对细胞存活至关重要，但错误的修复会引发基因组不稳定性（genetic instability），因此双链DNA断裂修复路径的选择是关键步骤。
本研究发现，位于双链DNA断裂位点附近的反向重复序列（inverted DNA repeats，IRs）可将原本通过精准路径——基因转换（gene conversion）——进行的断裂修复，引导至断裂诱导复制（break-induced replication，BIR）路径，进而引发基因组不稳定。
反向重复序列的这一效应，源于其在双链DNA断裂切除过程中形成的单链DNA（single-stranded DNA，ssDNA）中能够形成异常DNA结构的能力。
本研究证实，反向重复序列可形成两类异常DNA结构，二者的选择取决于分隔两个反向重复序列的间隔序列长度。
具体而言，带有长间隔序列（1 kb）的反向重复序列，主要通过分子间单链退火（inter-molecular single-strand annealing，SSA）路径形成反向二聚体；而带有短间隔序列（12 bp）的反向重复序列，则通过分子内单链退火（intra-molecular single-strand annealing，SSA）参与形成折返结构（fold-back，FB）。
这两类异常结构均会干扰通过基因转换进行的精准双链DNA断裂修复，并将修复路径引导至断裂诱导复制，进而促进基因组不稳定。
本研究同时发现，处理带有短（12 bp）与长（1 kb）单链DNA环的折返结构时，所需的蛋白质复合物存在差异。
具体而言，带有短单链DNA环的折返结构由MRX-Sae2复合物负责加工，而带有长单链DNA环的折返结构则由Rad1-Rad10复合物介导加工。
综上，本研究揭示了反向重复序列通过重定向双链DNA断裂修复至异常路径，进而引发基因组不稳定的具体机制。
鉴于人类基因组中反向重复序列分布广泛，本研究结果有助于理解由反向重复序列介导的、与人类疾病相关的基因组不稳定现象。