Plosky Brian S, Vidal Antonio E, Fernández de Henestrosa Antonio R, McLenigan Mary P, McDonald John P, Mead Samantha, Woodgate Roger
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2725, USA.
EMBO J. 2006 Jun 21;25(12):2847-55. doi: 10.1038/sj.emboj.7601178. Epub 2006 Jun 8.
Y-family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error-prone than high-fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer-termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Mutants of poliota (P692R) and poleta (H654A) were isolated that are defective in their interactions with polyUb and Ub-PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.
Y家族DNA聚合酶具有宽敞的活性位点，能够容纳各种各样的几何畸变。因此，它们比高保真复制酶更容易出错。所以，这些聚合酶在体内的活性受到严格调控，以尽量减少它们意外接近引物末端，这一点不足为奇。我们在此报告，人类细胞采用的一种此类机制依赖于DNA聚合酶ι和η与泛素（Ub）之间特定且直接的相互作用。实际上，我们表明这两种聚合酶都能与游离的多聚泛素链以及单泛素化的增殖细胞核抗原（Ub-PCNA）非共价相互作用。分离出了聚合酶ι（P692R）和聚合酶η（H654A）的突变体，它们在与多聚泛素和Ub-PCNA的相互作用方面存在缺陷，但仍保留与未修饰的PCNA相互作用的能力。有趣的是，这些聚合酶突变体在响应DNA损伤时表现出显著更低水平的复制灶，从而突出了聚合酶与泛素相互作用在体内调节跨损伤合成聚合酶接近停滞复制叉方面的生物学重要性。
