Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

中文翻译：

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PML同工型表达和相对于PML核体的DNA断裂位置影响同源重组的效率。

早幼粒细胞白血病核体（PML NBs）是核亚域，通过染色质结构的改变而数量增加，从而对遗传毒性胁迫作出反应。但是，尚未完全表征PML蛋白和PML NB在DNA修复的特定机制中的作用。在这里，我们已经使用I-SceI染色体外和基于染色体的同源指导的修复（HDR）分析直接检查了PML在同源重组（HR）中的作用，以及通过CRISPR / Cas9介导的基因编辑在HDR中的作用。我们确定PML丢失可以在染色体外HDR分析中抑制HR，但对CRISPR / Cas9介导的染色体HDR影响较小。PML的过表达还使用染色体报告基因并以同工型特异性方式抑制CRISPR HDR和I-SceI诱导的HDR。然而，PML过表达对染色体HDR报告基因的影响取决于报告基因在核内的染色体位置。具体而言，与不与PML NB相关的基因座相比，与PML NB相关的TAP1基因基因座处的HDR降低。但是，PML过度表达可在非PML NB相关基因座降低HDR。因此，PML同工型的丢失和过度表达均可以抑制HDR，并且染色体断裂与PML NB的接近可以影响HDR效率。