Trasviña-Arenas Carlos H, Cardona-Felix Cesar S, Azuara-Liceaga Elisa, Díaz-Quezada Corina, Brieba Luis G
Laboratorio Nacional de Genómica para la Biodiversidad Centro de Investigación y de Estudios Avanzados Irapuato Guanajuato México.
Present address: Centro Interdisciplinario de Ciencias Marinas (CICIMAR-IPN) Av. Instituto Politécnico Nacional. s/n.La Paz Baja California Sur 23096 Mexico.
FEBS Open Bio. 2017 Mar 16;7(5):659-674. doi: 10.1002/2211-5463.12209. eCollection 2017 May.
Proliferating cell nuclear antigen (PCNA) coordinates multienzymatic reactions by interacting with a variety of protein partners. Family I DNA ligases are multidomain proteins involved in sealing of DNA nicks during Okazaki fragment maturation and DNA repair. The interaction of DNA ligases with the interdomain connector loop (IDCL) of PCNA through its PCNA-interacting peptide (PIP box) is well studied but the role of the interacting surface between both proteins is not well characterized. In this work, we used a minimal DNA ligase I and two N-terminal deletions to establish that DNA binding and nick-sealing stimulation of DNA ligase I by PCNA are not solely dependent on the PIP box-IDCL interaction. We found that a truncated DNA ligase I with a deleted PIP box is stimulated by PCNA. Furthermore, the activity of a DNA ligase defective in DNA binding is rescued upon PCNA addition. As the rate constants for single-turnover ligation for the full-length and truncated DNA ligases are not affected by PCNA, our data suggest that PCNA stimulation is achieved by increasing the affinity for nicked DNA substrate and not by increasing catalytic efficiency. Surprisingly C-terminal mutants of PCNA are not able to stimulate nick-sealing activity of DNA ligase I. Our data support the notion that the C-terminal region of PCNA may be involved in promoting an allosteric transition in DNA ligase I from a spread-shaped to a ring-shaped structure. This study suggests that the ring-shaped PCNA is a binding platform able to stabilize coevolved protein-protein interactions, in this case an interaction with DNA ligase I.
增殖细胞核抗原（PCNA）通过与多种蛋白质伙伴相互作用来协调多酶反应。I 型 DNA 连接酶是多结构域蛋白质，参与冈崎片段成熟和 DNA 修复过程中 DNA 切口的封闭。DNA 连接酶通过其 PCNA 相互作用肽（PIP 框）与 PCNA 的结构域间连接环（IDCL）的相互作用已得到充分研究，但两种蛋白质之间相互作用表面的作用尚未得到很好的表征。在这项工作中，我们使用了最小化的 DNA 连接酶 I 和两个 N 端缺失来确定 PCNA 对 DNA 连接酶 I 的 DNA 结合和切口封闭刺激并不完全依赖于 PIP 框-IDCL 相互作用。我们发现缺失 PIP 框的截短 DNA 连接酶 I 受到 PCNA 的刺激。此外，添加 PCNA 后可挽救 DNA 结合缺陷的 DNA 连接酶的活性。由于全长和截短 DNA 连接酶的单轮连接速率常数不受 PCNA 的影响，我们的数据表明 PCNA 的刺激是通过增加对带切口 DNA 底物的亲和力而不是通过提高催化效率来实现的。令人惊讶的是，PCNA 的 C 端突变体无法刺激 DNA 连接酶 I 的切口封闭活性。我们的数据支持这样一种观点，即 PCNA 的 C 端区域可能参与促进 DNA 连接酶 I 从伸展形到环形结构的变构转变。这项研究表明，环形 PCNA 是一个结合平台，能够稳定共同进化的蛋白质-蛋白质相互作用，在这种情况下是与 DNA 连接酶 I 的相互作用。
