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Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

ABSTRACT: Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB.

SUBMITTER: Badrinarayanan A

PROVIDER: S-EPMC4523614 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

Badrinarayanan Anjana A Le Tung B K TB Laub Michael T MT

The Journal of cell biology 20150801 3

Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapid ...[more]

PMID: 26240183

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Project description: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. 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 ?M 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 ?M 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)

Dataset Information

Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

Publications

Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

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