title: The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity.
abstract: DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Shoujin
authors.initials: S
authors.lastname: Gu
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Li
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Hongtai
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Joy
authors.initials: J
authors.lastname: Fleming
authors.affiliation: School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Weiqiang
authors.initials: W
authors.lastname: Yang
authors.affiliation: School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Shihua
authors.initials: S
authors.lastname: Wang
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Wei
authors.affiliation: The Fourth People's Hospital, Foshan 528000, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China.
authors.forename: Guofeng
authors.initials: G
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Jiaoyu
authors.initials: J
authors.lastname: Deng
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Hou
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.|School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Shiqiang
authors.initials: S
authors.lastname: Lin
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.affiliation: Key Laboratory of RNA Biology &National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Bi
mesh_terms: D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D058105:Polymerization; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004269:DNA, Bacterial; D021122:Protein Subunits; C023140:DNA replicase; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases
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