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Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

Hu S, Wang J, Wang L, Zhang CC, Chen WL - PLoS ONE (2015)

Bottom Line: Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position.RecN-GFP was absent in most mature heterocysts.Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 430070 Wuhan, China.

ABSTRACT
DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

No MeSH data available.


Related in: MedlinePlus

RecN focus is close to the center of the cell treated with MMC.(A) Most RecN-GFP foci are colocalized with nucleoid at the center area of cells when treated with 2 μg/mL MMC. (B, C) RecN-GFP foci and nucleoids were colocalized at the position of cell plate between two daughter cells in dividing cell pairs. (D) RecN-GFP focus was localized in one of the daughter cells when the chromosome was segregated at the later period of cell cycle after MMC treatment. (E) The white arrows indicated that in a few cells, RecN–GFP foci were not colocalized with the chromosome at the central areas (5% in total, n = 196). (F) Nucleoids were more compacted and the number of RecN-GFP foci increased in some cells (white arrows) (21% of cell in a total, n = 213) after the treatment with 4 μg/mL MMC. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). Each photograph involved 3 different channels for DAPI (in blue) GFP (in green) fluorescence and bright field. Scale bars correspond to 1 μm.
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pone.0139362.g004: RecN focus is close to the center of the cell treated with MMC.(A) Most RecN-GFP foci are colocalized with nucleoid at the center area of cells when treated with 2 μg/mL MMC. (B, C) RecN-GFP foci and nucleoids were colocalized at the position of cell plate between two daughter cells in dividing cell pairs. (D) RecN-GFP focus was localized in one of the daughter cells when the chromosome was segregated at the later period of cell cycle after MMC treatment. (E) The white arrows indicated that in a few cells, RecN–GFP foci were not colocalized with the chromosome at the central areas (5% in total, n = 196). (F) Nucleoids were more compacted and the number of RecN-GFP foci increased in some cells (white arrows) (21% of cell in a total, n = 213) after the treatment with 4 μg/mL MMC. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). Each photograph involved 3 different channels for DAPI (in blue) GFP (in green) fluorescence and bright field. Scale bars correspond to 1 μm.

Mentions: If the role of RecN was to repair DSBs, its subcellular localization would be affected by occurrence of DNA lesions. To test and verify this possibility, we determined the subcellular localization of RecN–GFP after the treatment with 2 μg/mL MMC. We found that the majority of the RecN-GFP foci became constantly colocalized with nucleoids at the center of the cells (Fig 4A and S4C Fig). Even in dividing cell pairs, RecN-GFP foci and DNA were colocalized at the position of cell division plane (Fig 4B and 4C). Only in 5% (n = 196) cells, RecN–GFP foci were not colocalized with nucleoids in the central area (Fig 4E), but at the edge of nucleoid. After the chromosome was segregated into two daughter cells, the RecN-GFP focus of the mother cell would be inherited by one of the daughter cells (Fig 4D). The result was similar to that in untreated cells. Furthermore, in 21% (n = 213) of the cells, more than one RecN-GFP focus was found after treatment with MMC at 4 μg/mL (Fig 4F). MMC generates one-ended DSBs related to DNA replication. In contrast, when the cells were treated with nalidixic acid, which inhibited DNA gyrase activity and generated two-ended DSBs, chromosome DNA appeared to be more compacted as a result of the treatment; but few RecN-GFP foci were located at the cell center (S4A and S4C Fig) or at the position of cell division plane in dividing cells(S4B Fig).


Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

Hu S, Wang J, Wang L, Zhang CC, Chen WL - PLoS ONE (2015)

RecN focus is close to the center of the cell treated with MMC.(A) Most RecN-GFP foci are colocalized with nucleoid at the center area of cells when treated with 2 μg/mL MMC. (B, C) RecN-GFP foci and nucleoids were colocalized at the position of cell plate between two daughter cells in dividing cell pairs. (D) RecN-GFP focus was localized in one of the daughter cells when the chromosome was segregated at the later period of cell cycle after MMC treatment. (E) The white arrows indicated that in a few cells, RecN–GFP foci were not colocalized with the chromosome at the central areas (5% in total, n = 196). (F) Nucleoids were more compacted and the number of RecN-GFP foci increased in some cells (white arrows) (21% of cell in a total, n = 213) after the treatment with 4 μg/mL MMC. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). Each photograph involved 3 different channels for DAPI (in blue) GFP (in green) fluorescence and bright field. Scale bars correspond to 1 μm.
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Related In: Results  -  Collection

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pone.0139362.g004: RecN focus is close to the center of the cell treated with MMC.(A) Most RecN-GFP foci are colocalized with nucleoid at the center area of cells when treated with 2 μg/mL MMC. (B, C) RecN-GFP foci and nucleoids were colocalized at the position of cell plate between two daughter cells in dividing cell pairs. (D) RecN-GFP focus was localized in one of the daughter cells when the chromosome was segregated at the later period of cell cycle after MMC treatment. (E) The white arrows indicated that in a few cells, RecN–GFP foci were not colocalized with the chromosome at the central areas (5% in total, n = 196). (F) Nucleoids were more compacted and the number of RecN-GFP foci increased in some cells (white arrows) (21% of cell in a total, n = 213) after the treatment with 4 μg/mL MMC. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). Each photograph involved 3 different channels for DAPI (in blue) GFP (in green) fluorescence and bright field. Scale bars correspond to 1 μm.
Mentions: If the role of RecN was to repair DSBs, its subcellular localization would be affected by occurrence of DNA lesions. To test and verify this possibility, we determined the subcellular localization of RecN–GFP after the treatment with 2 μg/mL MMC. We found that the majority of the RecN-GFP foci became constantly colocalized with nucleoids at the center of the cells (Fig 4A and S4C Fig). Even in dividing cell pairs, RecN-GFP foci and DNA were colocalized at the position of cell division plane (Fig 4B and 4C). Only in 5% (n = 196) cells, RecN–GFP foci were not colocalized with nucleoids in the central area (Fig 4E), but at the edge of nucleoid. After the chromosome was segregated into two daughter cells, the RecN-GFP focus of the mother cell would be inherited by one of the daughter cells (Fig 4D). The result was similar to that in untreated cells. Furthermore, in 21% (n = 213) of the cells, more than one RecN-GFP focus was found after treatment with MMC at 4 μg/mL (Fig 4F). MMC generates one-ended DSBs related to DNA replication. In contrast, when the cells were treated with nalidixic acid, which inhibited DNA gyrase activity and generated two-ended DSBs, chromosome DNA appeared to be more compacted as a result of the treatment; but few RecN-GFP foci were located at the cell center (S4A and S4C Fig) or at the position of cell division plane in dividing cells(S4B Fig).

Bottom Line: Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position.RecN-GFP was absent in most mature heterocysts.Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 430070 Wuhan, China.

ABSTRACT
DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

No MeSH data available.


Related in: MedlinePlus