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| Status |
Public on Jul 03, 2015 |
| Title |
Rapid pairing and subsequent resegregation of distant homologous loci enables double-strand break repair in bacteria |
| Organism |
Caulobacter vibrioides NA1000 |
| Experiment type |
Other
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| Summary |
Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Consequently, cells in all domains of life have evolved mechanisms to repair DSBs, including through homologous recombination. Although recombination has been well characterized, the spatial organization of this process in living cells remains poorly understood. Here, we introduced site-specific DSBs in Caulobacter crescentus, and then used time-lapse microscopy to visualize the homology search, DSB repair, and the resegregation of chromosomal DNA. Even loci tethered to opposite cell poles can efficiently release, pair to enable recombination-based repair, and then resegregate to their original locations. Resegregation occurs independent of DNA replication and without disrupting global chromosome organization. Origin-proximal regions are resegregated by the same machinery, ParABS, used to segregate undamaged chromosomes following DNA replication. In contrast, origin-distal regions efficiently resegregate after a DSB independent of ParABS, and likely without dedicated segregation proteins. Instead, we propose that a physical, spring-like force drives the resegregation of origin-distal loci after DSB repair.
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| Overall design |
Caulobacter cells were depleted of DnaA for 1.5 h before synchronization. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and double-strand breaks were induced for 1 h by the addition of 500 μM vanillate. For control sample, no vanillate was added. Formadehyde (Sigma) was then added to the final concentration of 1%. Formadehyde crosslinks protein-DNA and DNA-DNA together, thereby capturing the structure of the chromosome at the time of fixation. Fixation was performed at the cell density of OD600 = 0.2. The crosslinking reactions were allowed to proceed for 30 minutes at 25 °C before quenching with 2.5 M glycine at a final concentration of 0.125 M. Fixed cells were then pelleted by centrifugation and subsequently washed twice with 1x M2 buffer (6.1 mM Na2HPO4, 3.9 mM KH2PO4, 9.3 mM NH4Cl, 0.5 mM MgSO4, 10 μM FeSO4, 0.5 mM CaCl2) before resuspending in 1x TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) to a final concentration of 107 cells per µl. Resuspended cells were then divided into 25 µl aliquots and stored at -80 °C for no more than 2 weeks. Each Hi-C experiment was performed using two of the 25 µL aliquots. Chromosome conformation capture with next-generation seqeuncing (Hi-C) was carried out exactly as described previously (Le et al., 2013 PMID: 24158908)
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| Contributor(s) |
Badrinarayanan A, Le TB, Laub MT |
| Citation(s) |
26240183 |
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| Submission date |
Mar 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Tung Ba Khanh Le |
| E-mail(s) |
tung.le@jic.ac.uk
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| Phone |
01603450776
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| Organization name |
John Innes Centre
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| Department |
Department of Molecular Microbiology
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| Lab |
www.tunglelab.org
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| Street address |
Colney Lane
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| City |
Norwich |
| State/province |
Norfolk |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL18276 |
Illumina HiSeq 2000 (Caulobacter crescentus NA1000) |
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| Samples (2) |
| GSM1632602 |
Laublab_BglII_HiC_NA1000_IsceI_site_33kb_from_oriC_IsceI_enzyme_induced |
| GSM1632603 |
Laublab_BglII_HiC_NA1000_IsceI_enzyme_absence |
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| Relations |
| BioProject |
PRJNA278008 |
| SRA |
SRP056099 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE66811_RAW.tar |
310.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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