Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide sequencing approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis, and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR sub-pathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology, and results in sequence insertion without additional break or SV. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Pold-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.结构基因组变异（SVs）的间断爆发式发生已在多种生物中被报道，但其成因仍未完全阐明。同源重组（HR）是一种以模板为导向的修复机制，可修复DNA双链断裂以及停滞或崩溃的复制叉。本团队近期发现了一条DNA断裂扩增与基因组重排通路，其起源于同源重组过程中形成的多入侵（MI）DNA联合分子的核酸内切酶加工过程。全基因组测序手段证实，多入侵诱导重排（MIR）常引发多种重复序列介导的结构基因组变异与非整倍体。本研究通过分子与遗传学分析，以及一种用于染色体重排定量的新型高灵敏度邻近连接检测法，进一步明确了两条MIR亚通路。MIR1是一种通用通路，可在任意序列环境中发生，会产生次级断裂并常引发额外的结构基因组变异。MIR2仅当重组供体存在显著同源性时才会激活，其会导致序列插入且不会产生额外断裂或结构基因组变异。与重组DNA合成不同，危害最强的MIR1通路会在部分持续存在的DNA联合分子上较晚发生，且不依赖PCNA/Pold。本研究深化了对这类基于同源重组的结构基因组变异形成通路的机制性认知，并证实复杂重复序列介导的结构基因组变异可在不发生位移DNA合成的情况下产生。本研究还提出了可从长读长测序数据中推断MIR1的序列特征。