In translesion synthesis (TLS), specialized DNA polymerases, such as polymerase (pol) η and Rev1, are recruited to stalled replication forks. These polymerases form a multi-protein complex with PCNA, Rad6-Rad18, and other specialized polymerases. Pol η interacts with PCNA and Rev1 via a PCNA-interacting protein (PIP) motif in its C-terminal unstructured region. Here we report the discovery of a second PIP-like motif in the C-terminal region of pol η, which we have designated as PIP2. We have designated the original PIP motif as PIP1. We show that the pol η PIP1 and PIP2 motifs bind PCNA with different affinities and kinetics. PIP1 binds with higher affinity than does PIP2, and PIP1 dissociates more slowly than does PIP2. In addition, we show that the interaction between pol η and Rad6-Rad18 is also mediated by the pol η PIP1 and PIP2 motifs. Again, we show that the affinity and kinetics by which these motifs bind Rad6-Rad18 is different. These findings are significant, because the multiple PIP-like motifs on pol η likely play quite different roles within the multi-protein complex formed at stalled replication forks. PIP1 likely plays a critical role in the recruiting pol η to this multi-protein complex. PIP2, by contrast, likely plays a critical role in maintaining the architecture and the dynamics of this multi-protein complex needed to maximize the efficiency and accuracy of TLS.

在跨损伤合成 (TLS) 中，专门的 DNA 聚合酶，如聚合酶 (pol) η 和 Rev1，被募集到停滞的复制叉。这些聚合酶与 PCNA、Rad6-Rad18 和其他专门的聚合酶形成多蛋白复合物。POLη与PCNA和修订版1相互作用经由位于其 C 端非结构化区域的 PCNA 相互作用蛋白 (PIP) 基序。在这里，我们报告在 pol η 的 C 端区域发现了第二个类似 PIP 的基序，我们将其指定为 PIP2。我们已将原始 PIP 主题指定为 PIP1。我们表明 pol η PIP1 和 PIP2 基序以不同的亲和力和动力学结合 PCNA。PIP1 以比 PIP2 更高的亲和力结合，并且 PIP1 比 PIP2 解离得更慢。此外，我们表明 pol η 和 Rad6-Rad18 之间的相互作用也由 pol η PIP1 和 PIP2 基序介导。我们再次表明，这些基序结合 Rad6-Rad18 的亲和力和动力学是不同的。这些发现很重要，因为 pol η 上的多个 PIP 样基序可能在停滞复制叉处形成的多蛋白复合物中发挥着截然不同的作用。PIP1 可能在将 pol η 招募到这种多蛋白复合物方面发挥着关键作用。相比之下，PIP2 可能在维持这种多蛋白复合物的结构和动态方面发挥着关键作用，以最大限度地提高 TLS 的效率和准确性。