Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Trieste 34149, Italy.
Institute of Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain.
Nucleic Acids Res. 2018 Oct 12;46(18):9816-9828. doi: 10.1093/nar/gky723.
p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.
p15PAF 是一种致癌的无规卷曲蛋白，通过与人类滑动夹 PCNA 相互作用来调节 DNA 复制和损伤绕过。在没有 DNA 的情况下，p15PAF 通过与滑动表面接触的扩展 PIP 盒穿过 PCNA 环。在这里，我们探测了 p15PAF-PCNA-DNA 三元复合物的原子尺度结构。晶体学和 MD 模拟表明，当 p15PAF 占据 PCNA 三聚体的两个亚基时，环道内的 DNA 与未占据的亚基结合。无 DNA 存在和存在时，PCNA 结合的 p15PAF 的结构是不变的，溶液 NMR 证实 DNA 不会从环壁上置换 p15PAF。因此，p15PAF 减少了 PCNA 的可用滑动表面，并且可能在复制聚合酶 (pol δ) 合成时充当将 DNA 固定到夹上的带。然而，这种约束可能需要通过跨损伤合成聚合酶 (pol η) 来释放，以实现有效的 DNA 损伤绕过。相应地，我们的生化数据表明，p15PAF 会损害 pol η-PCNA 全酶对受损和正常 DNA 模板的引物合成。根据我们的发现，我们讨论了 p15PAF 在 DNA 复制和抑制 DNA 损伤绕过中的可能机制作用。
