Structural basis for the multi-activity factor Rad5 in replication stress tolerance

• Published in: Nature communications Vol. 12; no. 1; p. 321
• Main Authors: Shen, Miaomiao, Dhingra, Nalini, Wang, Quan, Cheng, Chen, Zhu, Songbiao, Tian, Xiaolin, Yu, Jun, Gong, Xiaoxin, Li, Xuzhichao, Zhang, Hongwei, Xu, Xin, Zhai, Liting, Xie, Min, Gao, Ying, Deng, Haiteng, He, Yongning, Niu, Hengyao, Zhao, Xiaolan, Xiang, Song
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.01.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/337/151/2356; 631/337/458/582; 631/45; 631/535/1266; Adaptation, Physiological; Biocatalysis; Cell survival; Cellular structure; Conserved Sequence; Crystal structure; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA biosynthesis; Domains; Eukaryotes; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Humanities and Social Sciences; Kluyveromyces - metabolism; Metabolism; multidisciplinary; Proliferating cell nuclear antigen; Proliferating Cell Nuclear Antigen - metabolism; Protein Binding; Protein Domains; Protein structure; Proteins; Replication; Saccharomyces cerevisiae - metabolism; Science; Science (multidisciplinary); Stress; Stress, Physiological; Ubiquitin; Ubiquitin - metabolism; Ubiquitin-protein ligase; Ubiquitination; Yeast; Yeasts
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-20538-w

The yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress tolerance and cell survival using their multiple activities, including ubiquitin ligase, replication fork remodeling and DNA lesion targeting activities. Here, we present the crystal structure of a nearly full-length Rad5 protein. The structure shows three distinct, but well-connected, domains required for Rad5’s activities. The spatial arrangement of these domains suggest that different domains can have autonomous activities but also undergo intrinsic coordination. Moreover, our structural, biochemical and cellular studies demonstrate that Rad5’s HIRAN domain mediates interactions with the DNA metabolism maestro factor PCNA and contributes to its poly-ubiquitination, binds to DNA and contributes to the Rad5-catalyzed replication fork regression, defining a new type of HIRAN domains with multiple activities. Our work provides a framework to understand how Rad5 integrates its various activities in replication stress tolerance. Rad5 is a hub connecting three replication stress tolerance pathways. Here, the authors present the 3.3 Å crystal structure of a N-terminal truncated K.lactis Rad5 construct that reveals the spatial arrangement of the HIRAN, Snf2 and RING domains and structure-guided in vitro and in vivo experiments reveal multiple activities of the yeast Rad5 HIRAN domain among them a role in binding PCNA and supporting its ubiquitination.