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Cryo-EM structure of DNA polymerase θ helicase domain in complex with inhibitor novobiocin

Hanbo Guo, YanXia Wang, Jun Mao, Huimin Zhao, Yuntong He, Yuandong Hu, Jing Li, Yujie Liu, Zheng Guan, Allen Guo, Xiaodan Ni, Fengying Zhang, View ORCID ProfileJie Heng
doi: https://doi.org/10.1101/2023.01.20.524915
Hanbo Guo
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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YanXia Wang
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Jun Mao
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Huimin Zhao
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Yuntong He
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Yuandong Hu
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Jing Li
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Yujie Liu
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Zheng Guan
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Allen Guo
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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Xiaodan Ni
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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  • For correspondence: nixiaodan@shuimubio.com zhangfengying@shuimubio.com hengjie@shuimubio.com
Fengying Zhang
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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  • For correspondence: nixiaodan@shuimubio.com zhangfengying@shuimubio.com hengjie@shuimubio.com
Jie Heng
1Shuimu BioSciences Ltd., Life Science Park, Changping District, Beijing 102206, China
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  • ORCID record for Jie Heng
  • For correspondence: nixiaodan@shuimubio.com zhangfengying@shuimubio.com hengjie@shuimubio.com
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Abstract

DNA double-strand breaks (DSBs) are highly toxic lesions that occur during the cellular metabolic process. DNA Polymerase theta (Polθ) is an error-prone polymerase that has been implicated in the repair of chromosome breaks, recovery of broken replication forks, and translesion synthesis. The inhibition of Polθ activity has been implicated in killing HR-deficient tumor cells in vitro and in vivo. We present the first biochemical evidence that the antibiotics novobiocin (NVB) noncompetitively inhibit ATP hydrolysis by the ATPase domain of the Polθ helicase domain (Polθ-HLD). We report the Cryo-EM structure of apo dimeric Polθ helicase domain (Polθ-HLD), and the first inhibitor occupied Polθ-HLD structure. Our structure identifies a non-canonical novobiocin binding pocket, distinct from the canonical site that partially overlaps with the ATP in the ATPase domain. Comparison with the homolog helicase Hel308-DNA duplex complex suggests that the novobiocin competitively binds to a triangle hub on the DNA translocation pathway and blocks the ssDNA binding and translocation. Furthermore, the first dimeric structure of Polθ-HLD also provides a structural framework for revealing the microhomology-mediated end-joining mechanism. Our results demonstrate that the inhibitor-occupied structure combined with rational, structure-based drug design will undoubtedly accelerate the discovery of potent inhibitors with better efficacy and target selectivity to human Polθ.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted January 21, 2023.
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Cryo-EM structure of DNA polymerase θ helicase domain in complex with inhibitor novobiocin
Hanbo Guo, YanXia Wang, Jun Mao, Huimin Zhao, Yuntong He, Yuandong Hu, Jing Li, Yujie Liu, Zheng Guan, Allen Guo, Xiaodan Ni, Fengying Zhang, Jie Heng
bioRxiv 2023.01.20.524915; doi: https://doi.org/10.1101/2023.01.20.524915
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Cryo-EM structure of DNA polymerase θ helicase domain in complex with inhibitor novobiocin
Hanbo Guo, YanXia Wang, Jun Mao, Huimin Zhao, Yuntong He, Yuandong Hu, Jing Li, Yujie Liu, Zheng Guan, Allen Guo, Xiaodan Ni, Fengying Zhang, Jie Heng
bioRxiv 2023.01.20.524915; doi: https://doi.org/10.1101/2023.01.20.524915

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