Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance
Abstract DNA polymerases (Pols) play important roles in the transmission of genetic information. Although the function and (de)regulation of Pols are linked to many human diseases, the key mechanism of 3′‐OH deprotonation and the PPi formation are not totally clear. In this work, a method is present...
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Wiley
2025-01-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202408112 |
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| author | Lihua Zhao Yang Xu Zhiheng Yang Wenzhe Liu Shichao Zhong Jingwei Bai Xuefeng Guo |
| author_facet | Lihua Zhao Yang Xu Zhiheng Yang Wenzhe Liu Shichao Zhong Jingwei Bai Xuefeng Guo |
| author_sort | Lihua Zhao |
| collection | DOAJ |
| description | Abstract DNA polymerases (Pols) play important roles in the transmission of genetic information. Although the function and (de)regulation of Pols are linked to many human diseases, the key mechanism of 3′‐OH deprotonation and the PPi formation are not totally clear. In this work, a method is presented to detect the full catalytic cycle of human Pol (hPol β) in graphene‐molecule‐graphene single‐molecule junctions. Real‐time in situ monitoring successfully revealed the spatial and temporal properties of the open and closed conformation states of hPol β, distinguishing the reaction states in the Pols catalytic cycle and unveiling 3′‐OH deprotonation and pyrophosphate (PPi) formation mechanism of hPol β. Proton inventory experiment demonstrated that the rate‐limiting step of PPi formation is deprotonation, which occurs before a reverse conformational change. Additionally, by detecting the acidity (pKa), it is found that MgA‐bound OH− acted as a general base and activated the nucleophile of 3′‐OH, and that acidic residue D190 or D192 coordinated with MgB as a proton donor to PPi. This work provides useful insights into a fundamental chemical reaction that impacts genome synthesis efficiency and Pol fidelity, which the discovery of Pol‐targeting drugs and design of artificial Pols for DNA synthetic applications are expected to accelerated. |
| format | Article |
| id | doaj-art-7c6bb4e7204949f8898dcf0896eb5fb6 |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj-art-7c6bb4e7204949f8898dcf0896eb5fb62025-01-13T15:29:43ZengWileyAdvanced Science2198-38442025-01-01122n/an/a10.1002/advs.202408112Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule ConductanceLihua Zhao0Yang Xu1Zhiheng Yang2Wenzhe Liu3Shichao Zhong4Jingwei Bai5Xuefeng Guo6Beijing National Laboratory for Molecular Sciences National Biomedical Imaging Center College of Chemistry and Molecular Engineering Peking University 292 Chengfu Road, Haidian District Beijing 100871 P. R. ChinaSchool of Pharmaceutical Sciences Tsinghua University Beijing 100093 P. R. ChinaBeijing National Laboratory for Molecular Sciences National Biomedical Imaging Center College of Chemistry and Molecular Engineering Peking University 292 Chengfu Road, Haidian District Beijing 100871 P. R. ChinaBeijing National Laboratory for Molecular Sciences National Biomedical Imaging Center College of Chemistry and Molecular Engineering Peking University 292 Chengfu Road, Haidian District Beijing 100871 P. R. ChinaBeijing National Laboratory for Molecular Sciences National Biomedical Imaging Center College of Chemistry and Molecular Engineering Peking University 292 Chengfu Road, Haidian District Beijing 100871 P. R. ChinaSchool of Pharmaceutical Sciences Tsinghua University Beijing 100093 P. R. ChinaBeijing National Laboratory for Molecular Sciences National Biomedical Imaging Center College of Chemistry and Molecular Engineering Peking University 292 Chengfu Road, Haidian District Beijing 100871 P. R. ChinaAbstract DNA polymerases (Pols) play important roles in the transmission of genetic information. Although the function and (de)regulation of Pols are linked to many human diseases, the key mechanism of 3′‐OH deprotonation and the PPi formation are not totally clear. In this work, a method is presented to detect the full catalytic cycle of human Pol (hPol β) in graphene‐molecule‐graphene single‐molecule junctions. Real‐time in situ monitoring successfully revealed the spatial and temporal properties of the open and closed conformation states of hPol β, distinguishing the reaction states in the Pols catalytic cycle and unveiling 3′‐OH deprotonation and pyrophosphate (PPi) formation mechanism of hPol β. Proton inventory experiment demonstrated that the rate‐limiting step of PPi formation is deprotonation, which occurs before a reverse conformational change. Additionally, by detecting the acidity (pKa), it is found that MgA‐bound OH− acted as a general base and activated the nucleophile of 3′‐OH, and that acidic residue D190 or D192 coordinated with MgB as a proton donor to PPi. This work provides useful insights into a fundamental chemical reaction that impacts genome synthesis efficiency and Pol fidelity, which the discovery of Pol‐targeting drugs and design of artificial Pols for DNA synthetic applications are expected to accelerated.https://doi.org/10.1002/advs.202408112catalysis mechanismDNA polymeraseelectric fieldGMG biosensorhPol β proteinsingle‐molecule level |
| spellingShingle | Lihua Zhao Yang Xu Zhiheng Yang Wenzhe Liu Shichao Zhong Jingwei Bai Xuefeng Guo Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance Advanced Science catalysis mechanism DNA polymerase electric field GMG biosensor hPol β protein single‐molecule level |
| title | Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance |
| title_full | Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance |
| title_fullStr | Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance |
| title_full_unstemmed | Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance |
| title_short | Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance |
| title_sort | unveiling the mechanism of deprotonation and proton transfer of dna polymerase catalysis via single molecule conductance |
| topic | catalysis mechanism DNA polymerase electric field GMG biosensor hPol β protein single‐molecule level |
| url | https://doi.org/10.1002/advs.202408112 |
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