Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, TX 77555-1061, USA.
Proc Natl Acad Sci U S A. 2010 Jun 8;107(23):10401-5. doi: 10.1073/pnas.1005492107. Epub 2010 May 24.
The Rad6-Rad18 mediated monoubiquitylation of proliferating cell nuclear antigen (PCNA) at lys 164 plays a crucial role in promoting the access of translesion synthesis (TLS) DNA polymerases (Pols) to PCNA in the replication fork stalled at a lesion site. Although a number of genetic and biochemical observations have provided strong evidence that TLS Pols bind PCNA at its interdomain connector loop (IDCL) via their PCNA-interacting protein (PIP) domain, a more recent proposal formulates that TLS Pols bind PCNA at two sites, to the IDCL via their PIP domain and to lys-164 linked ubiquitin (Ub) via their ubiquitin-binding domain. To ascertain the relative contributions of the PIP and Ub-binding zinc finger (UBZ) domains of human Poleta in TLS, we have determined whether the C-terminal truncations of hPoleta that contain the PIP1 domain but lack the UBZ and PIP2 domains can still function in TLS in human cells. Our observations that such C-terminally truncated proteins promote efficient TLS opposite a cis-syn TT dimer and confer a high degree of UV resistance to XPV cells provide unambiguous evidence that the binding of PCNA via its PIP domain is essential as well as sufficient for providing hPoleta the ability to carry out TLS in human cells.
Rad6-Rad18 介导的增殖细胞核抗原 (PCNA) 在赖氨酸 164 上的单泛素化在促进跨损伤合成 (TLS) DNA 聚合酶 (Pols) 进入复制叉中停滞在损伤部位的 PCNA 中起着至关重要的作用。尽管许多遗传和生化观察提供了强有力的证据表明，TLS Pols 通过其 PCNA 相互作用蛋白 (PIP) 结构域结合 PCNA 的 IDCL，但最近的一项提议表明，TLS Pols 通过其 PIP 结构域与 IDCL 结合，并通过其泛素结合结构域与连接赖氨酸 164 的泛素 (Ub) 结合。为了确定人 Poleta 的 PIP 和 Ub 结合锌指 (UBZ) 结构域在 TLS 中的相对贡献，我们确定了是否包含 PIP1 结构域但缺乏 UBZ 和 PIP2 结构域的 hPoleta 的 C 末端截断仍然可以在人类细胞中的 TLS 中发挥作用。我们的观察结果表明，这种 C 末端截断的蛋白质能够有效地促进顺式-顺式 TT 二聚体对面的 TLS，并赋予 XPV 细胞高度的耐紫外线能力，这提供了明确的证据，即通过其 PIP 结构域结合 PCNA 对于赋予 hPoleta 在人类细胞中进行 TLS 的能力是必要的也是充分的。
