Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
J Mol Biol. 2013 Sep 9;425(17):3091-105. doi: 10.1016/j.jmb.2013.05.029. Epub 2013 Jun 7.
Rev1 is a Y-family translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap filling. In the process of TLS, high-fidelity replicative DNA polymerases stalled by DNA damage are replaced by error-prone TLS enzymes responsible for the majority of mutagenesis in eukaryotic cells. The polymerase exchange that gains low-fidelity TLS polymerases access to DNA is mediated by their interactions with proliferating cell nuclear antigen (PCNA). Rev1 stands alone from other Y-family TLS enzymes since it lacks the consensus PCNA-interacting protein box (PIP-box) motif, instead utilizing other modular domains for PCNA binding. Here we report solution NMR structure of an 11-kDa BRCA1 C-terminus (BRCT) domain from Saccharomyces cerevisiae Rev1 and demonstrate with the use of transverse relaxation optimized spectroscopy (TROSY) NMR methods that Rev1-BRCT domain directly interacts with an 87-kDa PCNA in solution. The domain adopts α/β fold (β1-α1-β2-β3-α2-β4-α3-α4) typical for BRCT domain superfamily. PCNA-binding interface of the Rev1-BRCT domain comprises conserved residues of the outer surface of the α1-helix and the α1-β1, β2-β3 and β3-α2 loops. On the other hand, Rev1-BRCT binds to the inter-domain region of PCNA that overlaps with the binding site for the PIP-box motif. Furthermore, Rev1-BRCT domain bound to PCNA can be displaced by increasing amounts of the PIP-box peptide from TLS DNA polymerase polη, suggesting that Rev1-BRCT and polη PIP-box interactions with the same PCNA monomer are mutually exclusive. These results provide structural insights into PCNA recognition by TLS DNA polymerases that help better understand TLS regulation in eukaryotes.
Rev1 是一种 Y 家族跨损伤合成（TLS）DNA 聚合酶，参与跨越 DNA 损伤部位的复制和复制后间隙填充。在 TLS 过程中，由 DNA 损伤引起的高保真复制 DNA 聚合酶失活，被负责真核细胞中大多数突变的易错 TLS 酶取代。获得低保真 TLS 聚合酶进入 DNA 的聚合酶交换由它们与增殖细胞核抗原（PCNA）的相互作用介导。Rev1 与其他 Y 家族 TLS 酶不同，因为它缺乏共识的 PCNA 相互作用蛋白盒（PIP-box）基序，而是利用其他模块域与 PCNA 结合。在这里，我们报告了来自酿酒酵母 Rev1 的 11kDa BRCA1 C 末端（BRCT）结构域的溶液 NMR 结构，并通过使用横向弛豫优化光谱（TROSY）NMR 方法证明 Rev1-BRCT 结构域在溶液中直接与 87kDa PCNA 相互作用。该结构域采用 α/β 折叠（β1-α1-β2-β3-α2-β4-α3-α4），这是 BRCT 结构域超家族的典型结构。Rev1-BRCT 结构域的 PCNA 结合界面包括 α1-螺旋和 α1-β1、β2-β3 和 β3-α2 环外表面的保守残基。另一方面，Rev1-BRCT 与 PCNA 结合的区域与 PIP-box 基序的结合位点重叠。此外，随着来自 TLS DNA 聚合酶 polη的 PIP-box 肽的增加，Rev1-BRCT 结构域与 PCNA 的结合可以被置换，这表明 Rev1-BRCT 和 polη PIP-box 与相同的 PCNA 单体的相互作用是相互排斥的。这些结果为 TLS DNA 聚合酶与 PCNA 的识别提供了结构见解，有助于更好地理解真核生物中的 TLS 调控。
