Warren Eric M, Vaithiyalingam Sivaraja, Haworth Justin, Greer Briana, Bielinsky Anja-Katrin, Chazin Walter J, Eichman Brandt F
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA.
Structure. 2008 Dec 10;16(12):1892-901. doi: 10.1016/j.str.2008.10.005.
Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.
Mcm10是真核生物DNA复制所必需的一种蛋白质，对复制叉的组装和进展至关重要。高度保守的内部结构域（Mcm10-ID）已被证明能与单链（ss）DNA、DNA聚合酶α和增殖细胞核抗原（PCNA）发生物理相互作用。此处展示的非洲爪蟾Mcm10-ID的晶体结构揭示了一种由寡核苷酸/寡糖折叠结构串联一个变体且高度碱性的锌指组成的DNA结合结构。体外DNA结合活性的核磁共振化学位移扰动和突变研究揭示了Mcm10如何利用这一独特表面与ssDNA结合。酿酒酵母中的相应突变导致对复制应激的敏感性增加，证明了Mcm10的这一区域与DNA结合对复制的功能重要性。此外，将已知破坏PCNA、聚合酶α和DNA相互作用的Mcm10突变映射到晶体结构上，有助于深入了解Mcm10如何在复制体中协调蛋白质与DNA的结合。
