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Structural Basis for the Multi-Activity Factor Rad5 in Replication Stress Tolerance

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Structural Basis for the Multi-Activity Factor Rad5 in Replication Stress Tolerance ARTICLE https://doi.org/10.1038/s41467-020-20538-w OPEN Structural basis for the multi-activity factor Rad5 in replication stress tolerance Miaomiao Shen 1,2,3, Nalini Dhingra4, Quan Wang 5, Chen Cheng6, Songbiao Zhu7, Xiaolin Tian7, Jun Yu8, Xiaoxin Gong1,2,3, Xuzhichao Li1,2,3, Hongwei Zhang8, Xin Xu 1,2,3, Liting Zhai8, Min Xie8, Ying Gao8, ✉ Haiteng Deng7, Yongning He6, Hengyao Niu5, Xiaolan Zhao 4 & Song Xiang 1,2,3 The yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress 1234567890():,; 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 coordina- tion. 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. 1 Department of Biochemistry and Molecular Biology, Tianjin Medical University, 300070 Tianjin, P. R. China. 2 Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Medical University, 300070 Tianjin, P. R. China. 3 The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, 300070 Tianjin, P. R. China. 4 Molecular Biology Department, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 5 Department of Molecular and Cellular Biochemistry, Indiana University Bloomington, Bloomington, IN 47405, USA. 6 State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, 201210 Shanghai, P. R. China. 7 MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systematic Biology, School of Life Sciences, Tsinghua University, 100084 Beijing, P. R. China. 8 CAS Key Laboratory of Nutrition, Metabolism and Food safety, Shanghai Institute of Nutrition and ✉ Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200031 Shanghai, P. R. China. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:321 | https://doi.org/10.1038/s41467-020-20538-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20538-w any types of genome lesions can impair the progression data indicate that distinct from the recently characterized HIRAN Mof DNA replication fork and hamper accurate genome domain in HLTF13–15, Rad5’s HIRAN domain possesses multiple duplication and cell survival. The multiple activities of activities that are critical for the Rad5-mediated processes. the budding yeast Rad5 protein help cells to cope with these disruptive events, which greatly increase during replication stress. Results Rad5 possesses a ubiquitin ligase activity and catalyzes the Lys63- The overall structure of the K. lactis Rad5. We screened through linked ubiquitin-chain modification of the proliferating cell a number of fungal species and were able to purify a nearly full- nuclear antigen (PCNA). This modification can trigger the error- length fragment of Rad5 from the yeast K. lactis (KlRad5, con- free branch of DNA damage tolerance (DDT), allowing DNA – taining residues 163–1114, Supplementary Fig. 2a, b) and crys- synthesis via template switch1 4. Rad5 also possesses ATP tallize it in the absence of DNA. KlRad5 shares 46.7% sequence hydrolysis-driven DNA translocase activity, through which it identity with ScRad5. The structure was determined using mer- catalyzes the regression of replication forks5. Replication fork cury anomalous diffraction signals from mercury-derivatized regression is a universal and regulated response to replication crystals. Structures of the mercury-derivatized and native crystals stresses in eukaryotes, which can lead to multiple consequences were refined to resolutions of 3.3 Å and 3.6 Å, respectively such as stabilizing the stalled replication fork and allowing lesion (Table 1). Minimal differences were observed between these bypass6,7. In addition, Rad5 recognizes stressed replication forks structures. In the remainder of the paper, we discuss the structure or DNA damage sites and recruits the translesion DNA poly- – of the mercury-derivatized crystal because of its higher resolution. merase Rev1 to these locations to promote DNA synthesis8 10. The structure revealed an elongated shape of KlRad5 spanning Orthologs of Rad5 have been identified in many eukaryotes. In 140 Å in the longest dimension (Fig. 1b). The monomeric crystal line with their critical roles in replication stress tolerance, the structure docks well into the density determined by negative human Rad5 orthologs, HLTF/HIP116 and SHPRH, are impli- staining electron microscopy (EM, Supplementary Fig. 3a–c) and cated in many types of cancer11,12. is consistent with a molecular weight of 120 kDa determined by A hallmark of the Rad5 family of proteins is the presence of dynamic light scattering, suggesting that KlRad5 adopts a multiple activity domains. The budding yeast S. cerevisiae Rad5 monomeric structure in a solution similar to the crystal and (ScRad5), a model for studying the Rad5 family of proteins, EM structures. The HIRAN, Snf2, and RING domains are well interacts with Rev1 through its N-terminal 30 residues8. The resolved in the structure. Each individual domain generally remaining part of ScRad5 contains three distinct domains, resembles previously reported homologous structures (Supple- including a HIP116 and Rad5 N-terminal (HIRAN) domain, a mentary Fig. 4a–d), but with important differences (detailed superfamily 2 (SF2) DNA translocase motor domain of the Snf2 below). The HIRAN domain and the two lobes in the Snf2 sub-family (Snf2 domain), and a ubiquitin ligase RING domain domain are nearly aligned in a line, the RING domain is located embedded in the Snf2 domain (Fig. 1a and Supplementary Fig. 1). on the side. Extensive interactions are observed between the Snf2 These distinct domains endow Rad5 with DNA damage site and HIRAN domains (Fig. 1c), and between the two lobes of the recognition9, replication fork remodeling5, and ubiquitin ligase1 Snf2 domain (Fig. 2a). In contrast, the RING domain has little activities. The assembly of multiple activity domains in contact with either the Snf2 or the HIRAN domain. Its active site Rad5 suggests complex functional mechanisms. Due to the cur- for ubiquitin transfer superimposes well with equivalent regions rent lack of structural information on the full-length proteins of in reported RING domain structures16 (Supplementary Fig. 4d–e) Rad5 or its orthologs, how they deploy distinct activities to fulfill and is fully solvent-exposed (Supplementary Fig. 4f). their multi-faceted role in replication stress tolerance is poorly understood. We present here a crystal structure of a nearly full-length Rad5 Structural basis for the Snf2 domain activation by DNA. SF2 protein. The structure and structure-guided functional studies enzymes hydrolyze ATP to drive nucleic acid translocation. They provide insights into the coordination of Rad5’s activities. Our contain seven conserved motifs distributed in two lobes. Fig. 1 Structure of KlRad5. a Domain organization of Rad5. The HIRAN and RING domains, the Snf2 domain lobes 1 and 2, and the mediator helix are indicated with different colors. The coloring scheme is used throughout the manuscript unless otherwise indicated. Domain boundaries for the K. latics Rad5 are indicated. b Crystal structure of KlRad5. Structural figures were prepared with PyMOL (http://pymol.org). c The mediator helix mediates interactions between the Snf2 and HIRAN domains. The β1–β3 insertion in the Snf2 domain lobe 1 is highlighted in red. Dashed lines indicate disordered regions in our structure. 2 NATURE COMMUNICATIONS | (2021) 12:321 | https://doi.org/10.1038/s41467-020-20538-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20538-w ARTICLE Table 1 Data collection and structure refinement statistics. DNA-binding cleft (Fig. 2c) and found that they caused strong reductions in KlRad5’saffinity to dsDNA (Fig. 2d) and its dsDNA-stimulated ATPase activity (Fig. 2e). The substituted Native crystal Mercury-derivatized crystal proteins behave similarly as the wild-type KlRad5 on a size- exclusion column (Supplementary Fig. 2a), suggesting that the Data collection substitutions did not affect the overall protein structure. Fourth, Space group P6222 P6222 Cell dimensions we introduced the E628A and Q1051D substitutions in KlRad5 a, b, c (Å) 188.54, 187.63, that are expected to disrupt the binding of the attacking water 17 188.54, 198.23 187.63, 197.54 molecule for ATP hydrolysis (Supplementary Fig. 5e), and α, β, γ (°) 90, 90, 120 90, 90, 120 found that they did not affect the overall protein folding Resolution (Å) 50.0–3.60 50.0–3.30 (Supplementary Fig. 2a) but severely inhibited the dsDNA- (3.66–3.60)* (3.36–3.30) stimulated ATPase activity (Fig. 2e). Fifth, the ATPase-inactive Rmerge 0.076 (1.406) 0.132 (3.566) conformation observed in our structure is stabilized by a I/σI 30.89 (1.53) 39.75 (1.80) hydrogen bond between Ser553 in the Snf2 domain lobe 1 and CC1/2 0.997 (0.524) 1.001 (0.548) the Glu953 side chain in lobe 2 (Fig. 2b), which is not expected to Completeness (%) 99.8 (99.0) 100.0 (100.0) interact with lobe 1 in the ATPase-active form (Supplementary Redundancy 6.9 (7.2) 56.1 (50.7) Fig. 5c). We removed this hydrogen bond by the E953A Refinement substitution and found that it did not affect the overall protein Resolution (Å) 50.0–3.60 50–3.30 (3.35–3.30) folding (Supplementary Fig.
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