Ducoux M, Urbach S, Baldacci G, Hübscher U, Koundrioukoff S, Christensen J, Hughes P
Institut Curie, UMR 2027 du CNRS, Génotoxicologie et Cycle Cellulaire, Bâtiment 110, Centre Universitaire, 91405 Orsay Cedex, France.
J Biol Chem. 2001 Dec 28;276(52):49258-66. doi: 10.1074/jbc.M106990200. Epub 2001 Oct 10.
The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.
介导增殖细胞核抗原(PCNA)与人DNA聚合酶δ结合的亚基尚未明确界定。我们发现,人DNA聚合酶δ的第三个亚基p66,通过位于其C末端的典型PCNA结合序列与PCNA相互作用。相反,p66与PCNA的结构域连接环相互作用,该区域先前已被证明对DNA聚合酶δ的活性以及细胞周期抑制剂p21(Cip1)的结合很重要。据此,含有p21(Cip1)的PCNA结合域的肽抑制了p66与PCNA的结合以及天然三亚基DNA聚合酶δ的活性。此外,下拉实验表明DNA聚合酶δ与PCNA相互作用需要p66。更重要的是,只有重组的三亚基DNA聚合酶δ显示出PCNA依赖的DNA复制,且该复制可被p21(Cip1)的PCNA结合域抑制。p66与PCNA和DNA聚合酶δ在DNA复制位点共定位,证明了p66在体内PCNA依赖的DNA复制中直接发挥作用。最后,细胞周期蛋白依赖性激酶对p66的体外磷酸化表明,p66的活性可能受到细胞周期依赖性调控。这些结果表明,p66是DNA聚合酶δ进行PCNA依赖的DNA合成的主要介导因子。