ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks
ABSTRACT RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed “threads,” which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP bi...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-12-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/msphere.00412-22 |
| _version_ | 1828113765491539968 |
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| author | Rogelio Hernández-Tamayo Niklas Steube Thomas Heimerl Georg K. A. Hochberg Peter L. Graumann |
| author_facet | Rogelio Hernández-Tamayo Niklas Steube Thomas Heimerl Georg K. A. Hochberg Peter L. Graumann |
| author_sort | Rogelio Hernández-Tamayo |
| collection | DOAJ |
| description | ABSTRACT RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed “threads,” which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, features lower dsDNA binding activity, but still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduced formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. In the presence of wild type RecA protein, additionally expressed RecA with lowered ATPbinding activity only moderately affected RecA dynamics, while loss of ATPase activity leads to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and nonspecifically DNA-bound molecules into threads upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Based on the idea that ATPase activity of RecA is most important for DNA strand exchange activity, our data suggest that extension and retraction of threads due is to many local strand invasion events during the search for sequences homologous to the induced DNA break site. IMPORTANCE Single-strand (ss) DNA binding ATPase RecA is the central recombinase in homologous recombination, and therefore essential for DNA repair pathways involving DNA strand exchange reactions. In several bacterial, RecA forms filamentous structures along the long axis of cells after induction of double strand breaks (DSBs) in the chromosome. These striking assemblies likely reflect RecA/ssDNA nucleoprotein filaments, which can extend and remodel within a time frame of few minutes. We show that ATPase activity of RecA is pivotal for these dynamic rearrangements, which include recruitment of freely diffusing molecules into low-mobile molecules within filaments. Our data suggest that ssDNA binding- and unbinding reactions are at the heart of RecA dynamics that power the dynamics of subcellular filamentous assemblies, leading to strand exchange reactions over a distance of several micrometers. |
| first_indexed | 2024-04-11T12:14:02Z |
| format | Article |
| id | doaj.art-946770b493494bbbb282a2a1360da27f |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-04-11T12:14:02Z |
| publishDate | 2022-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-946770b493494bbbb282a2a1360da27f2022-12-22T04:24:26ZengAmerican Society for MicrobiologymSphere2379-50422022-12-017610.1128/msphere.00412-22ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand BreaksRogelio Hernández-Tamayo0Niklas Steube1Thomas Heimerl2Georg K. A. Hochberg3Peter L. Graumann4Center for Synthetic Microbiology (SYNMIKRO), Marburg, GermanyCenter for Synthetic Microbiology (SYNMIKRO), Marburg, GermanyCenter for Synthetic Microbiology (SYNMIKRO), Marburg, GermanyCenter for Synthetic Microbiology (SYNMIKRO), Marburg, GermanyCenter for Synthetic Microbiology (SYNMIKRO), Marburg, GermanyABSTRACT RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed “threads,” which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, features lower dsDNA binding activity, but still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduced formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. In the presence of wild type RecA protein, additionally expressed RecA with lowered ATPbinding activity only moderately affected RecA dynamics, while loss of ATPase activity leads to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and nonspecifically DNA-bound molecules into threads upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Based on the idea that ATPase activity of RecA is most important for DNA strand exchange activity, our data suggest that extension and retraction of threads due is to many local strand invasion events during the search for sequences homologous to the induced DNA break site. IMPORTANCE Single-strand (ss) DNA binding ATPase RecA is the central recombinase in homologous recombination, and therefore essential for DNA repair pathways involving DNA strand exchange reactions. In several bacterial, RecA forms filamentous structures along the long axis of cells after induction of double strand breaks (DSBs) in the chromosome. These striking assemblies likely reflect RecA/ssDNA nucleoprotein filaments, which can extend and remodel within a time frame of few minutes. We show that ATPase activity of RecA is pivotal for these dynamic rearrangements, which include recruitment of freely diffusing molecules into low-mobile molecules within filaments. Our data suggest that ssDNA binding- and unbinding reactions are at the heart of RecA dynamics that power the dynamics of subcellular filamentous assemblies, leading to strand exchange reactions over a distance of several micrometers.https://journals.asm.org/doi/10.1128/msphere.00412-22homologous recombinationRecADNA break repairBacillus subtilisDNA repairdouble strand break repair |
| spellingShingle | Rogelio Hernández-Tamayo Niklas Steube Thomas Heimerl Georg K. A. Hochberg Peter L. Graumann ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks mSphere homologous recombination RecA DNA break repair Bacillus subtilis DNA repair double strand break repair |
| title | ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks |
| title_full | ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks |
| title_fullStr | ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks |
| title_full_unstemmed | ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks |
| title_short | ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks |
| title_sort | atpase activity of bacillus subtilis reca affects the dynamic formation of reca filaments at dna double strand breaks |
| topic | homologous recombination RecA DNA break repair Bacillus subtilis DNA repair double strand break repair |
| url | https://journals.asm.org/doi/10.1128/msphere.00412-22 |
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