Hexameric helicase G40P unwinds DNA in single base pair steps
Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G4...
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eLife Sciences Publications Ltd
2019-01-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/42001 |
| _version_ | 1818019675180630016 |
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| author | Michael Schlierf Ganggang Wang Xiaojiang S Chen Taekjip Ha |
| author_facet | Michael Schlierf Ganggang Wang Xiaojiang S Chen Taekjip Ha |
| author_sort | Michael Schlierf |
| collection | DOAJ |
| description | Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis. |
| first_indexed | 2024-04-14T07:55:27Z |
| format | Article |
| id | doaj.art-33fab9f87e804014ada8b475178f2bfd |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-14T07:55:27Z |
| publishDate | 2019-01-01 |
| publisher | eLife Sciences Publications Ltd |
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| spelling | doaj.art-33fab9f87e804014ada8b475178f2bfd2022-12-22T02:05:04ZengeLife Sciences Publications LtdeLife2050-084X2019-01-01810.7554/eLife.42001Hexameric helicase G40P unwinds DNA in single base pair stepsMichael Schlierf0https://orcid.org/0000-0002-6209-2364Ganggang Wang1Xiaojiang S Chen2https://orcid.org/0000-0001-9574-0551Taekjip Ha3https://orcid.org/0000-0003-2195-6258Physics Department and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Illinois, United States; B CUBE – Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, GermanyMolecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United StatesMolecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United StatesPhysics Department and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Illinois, United States; Howard Hughes Medical Institute, Baltimore, United States; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, United States; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States; Department of Biophysics, Johns Hopkins University, Baltimore, United StatesMost replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.https://elifesciences.org/articles/42001single-molecule FRETDnaBG40PDnaG |
| spellingShingle | Michael Schlierf Ganggang Wang Xiaojiang S Chen Taekjip Ha Hexameric helicase G40P unwinds DNA in single base pair steps eLife single-molecule FRET DnaB G40P DnaG |
| title | Hexameric helicase G40P unwinds DNA in single base pair steps |
| title_full | Hexameric helicase G40P unwinds DNA in single base pair steps |
| title_fullStr | Hexameric helicase G40P unwinds DNA in single base pair steps |
| title_full_unstemmed | Hexameric helicase G40P unwinds DNA in single base pair steps |
| title_short | Hexameric helicase G40P unwinds DNA in single base pair steps |
| title_sort | hexameric helicase g40p unwinds dna in single base pair steps |
| topic | single-molecule FRET DnaB G40P DnaG |
| url | https://elifesciences.org/articles/42001 |
| work_keys_str_mv | AT michaelschlierf hexamerichelicaseg40punwindsdnainsinglebasepairsteps AT ganggangwang hexamerichelicaseg40punwindsdnainsinglebasepairsteps AT xiaojiangschen hexamerichelicaseg40punwindsdnainsinglebasepairsteps AT taekjipha hexamerichelicaseg40punwindsdnainsinglebasepairsteps |