Department of Protein Chemistry, Leiden Institute of Chemistry, Gorlaeus Laboratory, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
DNA Repair (Amst). 2011 Sep 5;10(9):915-25. doi: 10.1016/j.dnarep.2011.04.033. Epub 2011 Jul 12.
Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C-terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein-protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5' phosphorylated, recessed, primer-template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5' primer-template junction binding, Rev1 efficiently binds to a recessed 3' primer-template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5' primer-template junctions, downstream of stalled replication forks.
Rev1 是一种真核生物 Y 家族 DNA 聚合酶，参与跨损伤合成（TLS），这是一种主要的损伤容忍途径，可允许在受损模板上进行 DNA 复制。在 Y 家族聚合酶中，Rev1 的 N 端部分（称为 BRCA1 C 端同源结构域（BRCT）区）具有独特的 BRCT 结构域。虽然大多数 BRCT 结构域介导蛋白质-蛋白质相互作用，但 Rev1 在 BRCT 结构域的 N 端包含一个预测的α-螺旋，在人类复制因子 C（RFC）中，这种 BRCT 结构域赋予蛋白质 DNA 结合能力。在这里，我们研究了酵母和小鼠 Rev1 的 DNA 结合特性。我们的结果表明，Rev1 的 BRCT 区域特异性结合到 5'磷酸化、凹陷的引物-模板连接点。这种 DNA 结合依赖于 BRCT 结构域 N 端的额外α-螺旋。令人惊讶的是，BRCT 结构域 N 端预测的α-螺旋之前的 20 个氨基酸也对高亲和力 DNA 结合至关重要。除了与 5'引物-模板连接点结合外，Rev1 还能有效地与凹陷的 3'引物-模板连接点结合。鉴于 Rev1 被停滞的复制叉下游的 5'引物-模板连接点招募，讨论了这些双重 DNA 结合特性。
