Mutual inhibition of RecQ molecules in DNA unwinding
Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2010 |
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| Erschienen: |
2010 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:285 |
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| Enthalten in: |
The Journal of biological chemistry - 285(2010), 21 vom: 21. Mai, Seite 15884-93 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Pan, Bing-Yi [VerfasserIn] |
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| Links: |
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| Themen: |
DNA, Bacterial |
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| Anmerkungen: |
Date Completed 14.06.2010 Date Revised 06.02.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1074/jbc.M110.104299 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM195834909 |
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| 245 | 1 | 0 | |a Mutual inhibition of RecQ molecules in DNA unwinding |
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a DNA, Bacterial |2 NLM | |
| 650 | 7 | |a Escherichia coli Proteins |2 NLM | |
| 650 | 7 | |a RecQ protein, E coli |2 NLM | |
| 650 | 7 | |a EC 3.6.1.- |2 NLM | |
| 650 | 7 | |a RecQ Helicases |2 NLM | |
| 650 | 7 | |a EC 3.6.4.12 |2 NLM | |
| 700 | 1 | |a Dou, Shuo-Xing |e verfasserin |4 aut | |
| 700 | 1 | |a Yang, Ye |e verfasserin |4 aut | |
| 700 | 1 | |a Xu, Ya-Nan |e verfasserin |4 aut | |
| 700 | 1 | |a Bugnard, Elisabeth |e verfasserin |4 aut | |
| 700 | 1 | |a Ding, Xiu-Yan |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Lingyun |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Peng-Ye |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Ming |e verfasserin |4 aut | |
| 700 | 1 | |a Xi, Xu Guang |e verfasserin |4 aut | |
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| 856 | 4 | 0 | |u http://dx.doi.org/10.1074/jbc.M110.104299 |3 Volltext |
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