Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ.
Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ.
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DOI：
10.1016/j.jbc.2022.102859
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发表时间：
2023-03
影响因子：
4.8
通讯作者：
Korzhnev, Dmitry M.
中科院分区：
文献类型：
--
作者：
McPherson, Kerry Silva;Rizzo, Alessandro A.;Erlandsen, Heidi;Chatterjee, Nimrat;Walker, Graham C.;Korzhnev, Dmitry M.
翻译合成（TLS）DNA聚合酶Polymerase对于DNA损伤位点上的旁路复制至关重要。Pol β的Rev 7亚基是HORMA（Hop 1、Rev 7、Mad 2）蛋白，其通过与催化亚基Rev 3和跨损伤合成支架蛋白Rev 1的相互作用促进Pol β向复制叉的募集。人Rev 7（hRev 7）通过HORMA蛋白中保守的机制与hRev 3的两个Rev 7结合基序（RBM）相互作用，由此hRev 7的安全带环在配体的顶部闭合。由两个hRev 3-RBM拴系的hRev 7的两个拷贝通过典型的HORMA二聚化界面形成对称的头对头二聚体。最近的冷冻-EM结构揭示了酿酒酵母Pol β（scPol β）还包括结合到scRev 3的不同区域的scRev 7的两个拷贝。令人惊讶的是，HORMA二聚化界面在scRev 7中不保守，两个scRev 7原聚体形成具有比hRev 7二聚体小得多的界面的不对称头-尾二聚体。在这里，我们验证了scRev 3中两个相邻的RBM基序，它们与scRev 7的结合亲和力相差两个数量级，并证实了溶液中scRev 7：Rev 3复合物的化学计量比为2：1。然而，我们的生物物理学研究表明，scRev 7在溶液中不形成二聚体，无论是在其自身的雅阁或当被scRev 3中的两个RBM束缚时。这些发现意味着在冷冻EM结构中观察到的scRev 7二聚体是由scRev 7与其他Pol γ亚基的相互作用诱导的，并且通过HORMA界面的Rev 7同源二聚化是在进化后期出现的机制。
Translesion synthesis (TLS) DNA polymerase Polζ is crucial for the bypass replication over sites of DNA damage. The Rev7 subunit of Polζ is a HORMA (Hop1, Rev7, Mad2) protein that facilitates recruitment of Polζ to the replication fork via interactions with the catalytic subunit Rev3 and the translesion synthesis scaffold protein Rev1. Human Rev7 (hRev7) interacts with two Rev7-binding motifs (RBMs) of hRev3 by a mechanism conserved among HORMA proteins whereby the safety-belt loop of hRev7 closes on the top of the ligand. The two copies of hRev7 tethered by the two hRev3-RBMs form a symmetric head-to-head dimer through the canonical HORMA dimerization interface. Recent cryo-EM structures reveal that Saccharomyces cerevisiae Polζ (scPolζ) also includes two copies of scRev7 bound to distinct regions of scRev3. Surprisingly, the HORMA dimerization interface is not conserved in scRev7, with the two scRev7 protomers forming an asymmetric head-to-tail dimer with a much smaller interface than the hRev7 dimer. Here, we validated the two adjacent RBM motifs in scRev3, which bind scRev7 with affinities that differ by two orders of magnitude and confirmed the 2:1 stoichiometry of the scRev7:Rev3 complex in solution. However, our biophysical studies reveal that scRev7 does not form dimers in solution either on its own accord or when tethered by the two RBMs in scRev3. These findings imply that the scRev7 dimer observed in the cryo-EM structures is induced by scRev7 interactions with other Polζ subunits and that Rev7 homodimerization via the HORMA interface is a mechanism that emerged later in evolution.
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