Kondratick Christine M, Boehm Elizabeth M, Dieckman Lynne M, Powers Kyle T, Sanchez Julio C, Mueting Samuel R, Washington M Todd
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
Department of Chemistry, Creighton University, Omaha, Nebraska, 68178, United States of America.
PLoS One. 2016 Jun 3;11(6):e0157023. doi: 10.1371/journal.pone.0157023. eCollection 2016.
Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.
增殖细胞核抗原（PCNA）通过与大量参与DNA复制和修复过程的蛋白质相互作用，在这些过程中发挥着至关重要的作用。PCNA中的两个氨基酸替换均位于亚基界面，先前已证明它们会阻断跨损伤合成（TLS），这是一种在复制过程中绕过DNA损伤的途径。为了更好地理解亚基界面在TLS中的作用，我们使用随机诱变产生了一组33个在亚基界面有替换的PCNA突变体。我们检测了这组突变体的全部活力以及对紫外线（UV）辐射的敏感性。然后，我们选择了17个突变体的子集，并测量了它们的细胞生长速率、自发诱变率和紫外线诱导的诱变率。这17个突变体中除了3个以外，其余的在诱导诱变方面都存在部分或完全缺陷，这表明TLS部分或完全丧失。这些结果表明，PCNA亚基界面的完整性对于高效的TLS至关重要，而且即使是保守替换也有可能破坏这一过程。
