Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE 19716, USA.
Nucleic Acids Res. 2021 Sep 20;49(16):9374-9388. doi: 10.1093/nar/gkab646.
The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.
Y 家族 DNA 聚合酶 η（Polη）在酵母中通过跨损伤 DNA 合成（TLS）对合成受损 DNA 核苷酸至关重要。TLS 是通过增殖细胞核抗原（PCNA）的单泛素化和随后的 TLS 聚合酶募集启动的。尽管已经解决了 Polη 催化核心和 PCNA 的个别结构，但 Polη/PCNA 或 Polη/单泛素化 PCNA（Ub-PCNA）复合物的高分辨率结构仍然难以捉摸，部分原因是 Polη C 端区域的无序性和 PCNA 上泛素的灵活性。为了克服这些障碍并深入了解这种重要的 TLS 聚合酶复合物，我们开发了含有 p-苯甲酰基-L-苯丙氨酸（pBpa）交联剂的光激活 PCNA 和 Ub-PCNA 探针，该交联剂位于 PCNA 上的选定位置。通过用光将探针与全长 Polη 交联，在胰蛋白酶消化和串联质谱分析后鉴定出特定的交联位点。我们发现 Polη 催化核心及其 C 端区域与 PCNA 环的两侧直接相互作用。使用交联位点信息作为约束的建模揭示了聚合酶复合物中 Polη 的多种构象。光激活 PCNA 和 Ub-PCNA 探针的可用性也将有助于研究对 DNA 复制、修复和损伤容忍至关重要的其他含有 PCNA 的复合物。
