Wilson Rosemary H C, Biasutto Antonio J, Wang Lihao, Fischer Roman, Baple Emma L, Crosby Andrew H, Mancini Erika J, Green Catherine M
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
DNA Repair (Amst). 2017 Feb;50:22-35. doi: 10.1016/j.dnarep.2016.12.003. Epub 2016 Dec 31.
Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3 and PolD4. In contrast p21 largely retains the ability to bind PCNA. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.
增殖细胞核抗原（PCNA）是DNA复制和修复过程中必不可少的辅助因子，可将多种蛋白质招募到其作用位点。我们研究了导致PCNA相关DNA修复障碍（PARD）的PCNA突变的影响。来自受PARD影响个体的细胞对PCNA抑制剂T3和T2AA敏感，这表明S228I突变对未受损细胞有影响。对PCNA与PCNA相互作用蛋白（PIPs）之间结合的分析表明，S228I变化极大地损害了这些相互作用中的大多数，包括Cdt1、DNMT1、PolD3和PolD4的相互作用。相比之下，p21在很大程度上保留了与PCNA结合的能力。这种特性由p21 PIP框序列本身赋予，该序列对于PCNA结合既必要又充分。PCNA的泛素化不受S228I变化的影响，S228I变化间接改变了结构域间连接环的结构。尽管PARD突变在体外对PIP-降解子结合有显著影响，但受影响个体细胞中p21和Cdt1的稳定性只有轻微改变。总体而言，我们的数据表明PCNA对特定客户的亲和力降低会在未受损细胞中导致细微的细胞缺陷，这可能有助于PARD的病因学研究。
