Molecular basis for PrimPol recruitment to replication forks by RPA.
Molecular basis for PrimPol recruitment to replication forks by RPA.
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DOI：
10.1038/ncomms15222
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发表时间：
2017-05-23
影响因子：
16.6
通讯作者：
Doherty AJ
中科院分区：
文献类型：
--
作者：
Guilliam TA;Brissett NC;Ehlinger A;Keen BA;Kolesar P;Taylor EM;Bailey LJ;Lindsay HD;Chazin WJ;Doherty AJ
DNA损伤和二级结构会使复制机制停滞。细胞在存在这些障碍的情况下具有许多耐受机制来完成基因组复制。除了翻译合成（TLS）聚合酶外，大多数真核细胞还含有一种称为引物聚合酶（PrimPol）的多功能复制酶，该酶能够直接绕过TLS的DNA损伤，并在障碍下游重新启动复制。在这里，我们报道PrimPol通过与RPA的相互作用被招募来重新启动。利用生物物理和晶体学方法，我们确定PrimPol具有两个rpa结合基序，并确定了这些相互作用所需的关键残基。我们证明了其中一个基序对于PrimPol的招募在体内阻止复制分叉是至关重要的。此外，生化分析表明RPA对PrimPol引物酶活性有刺激作用。总之，这些发现为PrimPol的招募模式提供了重要的分子见解，以促进重新启动和重新启动。PrimPol是一种多功能复制酶，可以绕过DNA损伤，并重新启动复制。在这里，作者阐明了PrimPol是如何通过与RPA的特定相互作用来阻止复制分叉的，RPA刺激其引物酶活性。
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase–polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart. PrimPol is a multifunctional replicative enzyme that can bypass DNA damage, as well as reprime replication restart. Here, the authors have elucidated how PrimPol is recruited to stalled replication forks via specific interactions with RPA, which stimulates its primase activity.