Chin Sang Christopher, Moore Gaelen, Tereshchenko Maria, Zhang Hongshan, Nosella Michael L, Dasovich Morgan, Alderson T Reid, Leung Anthony K L, Finkelstein Ilya J, Forman-Kay Julie D, Lee Hyun O
Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
EMBO Rep. 2024 Dec;25(12):5635-5666. doi: 10.1038/s44319-024-00285-5. Epub 2024 Nov 4.
Poly(ADP-ribose) polymerase 1 (PARP1) is one of the first responders to DNA damage and plays crucial roles in recruiting DNA repair proteins through its activity - poly(ADP-ribosyl)ation (PARylation). The enrichment of DNA repair proteins at sites of DNA damage has been described as the formation of a biomolecular condensate. However, it remains unclear how exactly PARP1 and PARylation contribute to the formation and organization of DNA repair condensates. Using recombinant human single-strand repair proteins in vitro, we find that PARP1 readily forms viscous biomolecular condensates in a DNA-dependent manner and that this depends on its three zinc finger (ZnF) domains. PARylation enhances PARP1 condensation in a PAR chain length-dependent manner and increases the internal dynamics of PARP1 condensates. DNA and single-strand break repair proteins XRCC1, LigIII, Polβ, and FUS partition in PARP1 condensates, although in different patterns. While Polβ and FUS are both homogeneously mixed within PARP1 condensates, FUS enrichment is greatly enhanced upon PARylation whereas Polβ partitioning is not. XRCC1 and LigIII display an inhomogeneous organization within PARP1 condensates; their enrichment in these multiphase condensates is enhanced by PARylation. Functionally, PARP1 condensates concentrate short DNA fragments, which correlates with PARP1 clusters compacting long DNA and bridging DNA ends. Furthermore, the presence of PARP1 condensates significantly promotes DNA ligation upon PARylation. These findings provide insight into how PARP1 condensation and PARylation regulate the assembly and biochemical activities of DNA repair factors, which may inform on how PARPs function in DNA repair foci and other PAR-driven condensates in cells.
聚（ADP - 核糖）聚合酶1（PARP1）是DNA损伤的首批响应者之一，通过其聚（ADP - 核糖基）化（PARylation）活性在招募DNA修复蛋白方面发挥关键作用。DNA损伤位点处DNA修复蛋白的富集被描述为生物分子凝聚物的形成。然而，PARP1和PARylation究竟如何促进DNA修复凝聚物的形成和组织仍不清楚。我们在体外使用重组人单链修复蛋白，发现PARP1以DNA依赖性方式容易形成粘性生物分子凝聚物，这取决于其三个锌指（ZnF）结构域。PARylation以PAR链长度依赖性方式增强PARP1凝聚，并增加PARP1凝聚物的内部动力学。DNA和单链断裂修复蛋白XRCC1、LigIII、Polβ和FUS在PARP1凝聚物中分配，尽管模式不同。虽然Polβ和FUS在PARP1凝聚物中均均匀混合，但PARylation后FUS的富集大大增强，而Polβ的分配则没有。XRCC1和LigIII在PARP1凝聚物中显示出不均匀的组织；PARylation增强了它们在这些多相凝聚物中的富集。在功能上，PARP1凝聚物浓缩短DNA片段，这与PARP1簇压缩长DNA和连接DNA末端相关。此外，PARP1凝聚物的存在显著促进PARylation后的DNA连接。这些发现深入了解了PARP1凝聚和PARylation如何调节DNA修复因子的组装和生化活性，这可能为PARP在细胞中DNA修复位点和其他PAR驱动的凝聚物中的功能提供信息。
