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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 appears as a single discrete globular focus in newly formed heterocyst and disappears in mature ones.(A) RecN-GFP focus in heterocyst (white arrow). (B) Rates (in percentage) of heterocysts with RecN–GFP foci after the starvation of combined nitrogen. (C) Western blotting analysis of protein extracts from vegetative cells or enriched heterocysts using anti-RecN antiserum. Lanes 1 and 2: extracts from vegetative cell in 0 and 12 h after nitrogen deprivation; Lanes 3, 4 and 5: extracts from heterocyst in 24, 48 and 72 h after nitrogen deprivation. (D) High concentration of MMC did not lead to the reformation of RecN-GFP foci in mature heterocysts. After 24 h deprivation of combined nitrogen, 4 μg/mL MMC was added into the medium for 3-day cultivation and then the photographs were captured. The white arrows indicate heterocysts. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). The red fluorescence is the fluorescence of photosynthetic pigment. Scale bars correspond to 1 μm.
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pone.0139362.g005: RecN appears as a single discrete globular focus in newly formed heterocyst and disappears in mature ones.(A) RecN-GFP focus in heterocyst (white arrow). (B) Rates (in percentage) of heterocysts with RecN–GFP foci after the starvation of combined nitrogen. (C) Western blotting analysis of protein extracts from vegetative cells or enriched heterocysts using anti-RecN antiserum. Lanes 1 and 2: extracts from vegetative cell in 0 and 12 h after nitrogen deprivation; Lanes 3, 4 and 5: extracts from heterocyst in 24, 48 and 72 h after nitrogen deprivation. (D) High concentration of MMC did not lead to the reformation of RecN-GFP foci in mature heterocysts. After 24 h deprivation of combined nitrogen, 4 μg/mL MMC was added into the medium for 3-day cultivation and then the photographs were captured. The white arrows indicate heterocysts. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). The red fluorescence is the fluorescence of photosynthetic pigment. Scale bars correspond to 1 μm.

Mentions: Heterocysts are terminally differentiated nitrogen-fixing cells for supplying fixed nitrogen for neighboring vegetative cells. We wonder whether the DNA repair of DSBs is still active in heterocysts because of their terminal nature after differentiation. We performed the subcellular localization of RecN–GFP in heterocysts and found that RecN was localized as a discrete focus in the similar manner to that in vegetative cells (Fig 5A). However, only some heterocysts had a RecN-GFP focus. In a prolonged time after the induction of heterocyst formation, the percentage of heterocysts with a RecN–GFP focus among all heterocysts dropped from 84.2% in 24 h to 26.3% in 120 h (Fig 5B). Consistent with these observations, western blotting also showed that the level of the RecN protein decreased steadily in purified heterocysts after the induction by the deprivation of combined nitrogen (Fig 5C). These observations indicated that a RecN–GFP focus was present in the early stage of heterocyst formation and disappeared overtime in mature heterocysts. Furthermore, 4 μg/mL MMC was added into the medium after 24 h nitrogen starvation, followed by 3-day cultivation. However, most heterocysts still did not display a RecN-GFP focus, in contrast to the results observed in vegetative cells (Fig 5D). DNA appeared more condensed after treatment by MMC in both vegetative cells and heterocysts. These results suggest that the DNA repair system for DSBs may be no longer functional in mature heterocysts.


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 appears as a single discrete globular focus in newly formed heterocyst and disappears in mature ones.(A) RecN-GFP focus in heterocyst (white arrow). (B) Rates (in percentage) of heterocysts with RecN–GFP foci after the starvation of combined nitrogen. (C) Western blotting analysis of protein extracts from vegetative cells or enriched heterocysts using anti-RecN antiserum. Lanes 1 and 2: extracts from vegetative cell in 0 and 12 h after nitrogen deprivation; Lanes 3, 4 and 5: extracts from heterocyst in 24, 48 and 72 h after nitrogen deprivation. (D) High concentration of MMC did not lead to the reformation of RecN-GFP foci in mature heterocysts. After 24 h deprivation of combined nitrogen, 4 μg/mL MMC was added into the medium for 3-day cultivation and then the photographs were captured. The white arrows indicate heterocysts. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). The red fluorescence is the fluorescence of photosynthetic pigment. Scale bars correspond to 1 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4592062&req=5

pone.0139362.g005: RecN appears as a single discrete globular focus in newly formed heterocyst and disappears in mature ones.(A) RecN-GFP focus in heterocyst (white arrow). (B) Rates (in percentage) of heterocysts with RecN–GFP foci after the starvation of combined nitrogen. (C) Western blotting analysis of protein extracts from vegetative cells or enriched heterocysts using anti-RecN antiserum. Lanes 1 and 2: extracts from vegetative cell in 0 and 12 h after nitrogen deprivation; Lanes 3, 4 and 5: extracts from heterocyst in 24, 48 and 72 h after nitrogen deprivation. (D) High concentration of MMC did not lead to the reformation of RecN-GFP foci in mature heterocysts. After 24 h deprivation of combined nitrogen, 4 μg/mL MMC was added into the medium for 3-day cultivation and then the photographs were captured. The white arrows indicate heterocysts. Photographs were taken by using an Olympus FV1000 confocal microscope. Cells were stained with DAPI (blue). The red fluorescence is the fluorescence of photosynthetic pigment. Scale bars correspond to 1 μm.
Mentions: Heterocysts are terminally differentiated nitrogen-fixing cells for supplying fixed nitrogen for neighboring vegetative cells. We wonder whether the DNA repair of DSBs is still active in heterocysts because of their terminal nature after differentiation. We performed the subcellular localization of RecN–GFP in heterocysts and found that RecN was localized as a discrete focus in the similar manner to that in vegetative cells (Fig 5A). However, only some heterocysts had a RecN-GFP focus. In a prolonged time after the induction of heterocyst formation, the percentage of heterocysts with a RecN–GFP focus among all heterocysts dropped from 84.2% in 24 h to 26.3% in 120 h (Fig 5B). Consistent with these observations, western blotting also showed that the level of the RecN protein decreased steadily in purified heterocysts after the induction by the deprivation of combined nitrogen (Fig 5C). These observations indicated that a RecN–GFP focus was present in the early stage of heterocyst formation and disappeared overtime in mature heterocysts. Furthermore, 4 μg/mL MMC was added into the medium after 24 h nitrogen starvation, followed by 3-day cultivation. However, most heterocysts still did not display a RecN-GFP focus, in contrast to the results observed in vegetative cells (Fig 5D). DNA appeared more condensed after treatment by MMC in both vegetative cells and heterocysts. These results suggest that the DNA repair system for DSBs may be no longer functional in mature heterocysts.

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