Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, 06030, United States.
Biochemistry. 2012 Jul 10;51(27):5506-20. doi: 10.1021/bi300566z. Epub 2012 Jun 28.
Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.
Rev1 是一种跨损伤合成（TLS）DNA 聚合酶，对于真核生物中的 DNA 损伤容忍至关重要。在 TLS 过程中，停滞不前的高保真复制 DNA 聚合酶会被专门的 TLS 酶暂时取代，这些酶可以绕过 DNA 损伤（损伤）部位，从而允许复制继续或填补复制后缺口。尽管人类 Rev1 的催化活性有限，但它在 TLS 中起着关键作用，作为一个支架，为 Y 家族 TLS 聚合酶 polη、ι 和 κ 提供了进入其同源 DNA 损伤的途径，并促进它们随后交换到 polζ，从而延伸扭曲的 DNA 引物-模板。Rev1 与其他主要的人类 TLS 聚合酶，polη、ι、κ 和 polζ 的调节亚基 Rev7 的相互作用是由 Rev1 C 端结构域（Rev1-CT）介导的。我们使用 NMR 光谱学来确定 Rev1-CT 结构域（残基 1157-1251）的空间结构及其与 polη（残基 524-539）的 Rev1 相互作用区域（RIR）的复合物。该结构域形成一个四螺旋束，具有结构良好的 N 端β发夹，与螺旋 1 和 2 对接，为 RIR 基序的两个保守苯丙氨酸残基创建一个结合口袋，当结合时，这些残基折叠成一个α螺旋。NMR 自旋松弛和 NMR 弛豫分散测量表明，游离 Rev1-CT 和 Rev1-CT/polη-RIR 复合物表现出μs-ms 构象动力学，涵盖 RIR 结合位点，这可能有助于选择最适合结合的分子构型。这些结果为理解 Rev1-CT 被 Y 家族 DNA 聚合酶识别提供了结构基础，为理解人类细胞中的 TLS 控制提供了新的见解。
