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Reduction of hRNase H2 activity in Aicardi-Goutières syndrome cells leads to replication stress and genome instability.

Pizzi S, Sertic S, Orcesi S, Cereda C, Bianchi M, Jackson AP, Lazzaro F, Plevani P, Muzi-Falconi M - Hum. Mol. Genet. (2014)

Bottom Line: Our data indicate that in human cells RNase H2 plays a crucial role in correcting rNMPs misincorporation, preventing DNA damage.Such protective function is compromised in AGS patients and may be linked to unscheduled immune responses.These findings may be relevant to shed further light on the mechanisms involved in AGS pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.

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RNase H2 depletion causes a delay in S-G2 phases. Cell cycle distribution of asynchronous HeLa cells. Histograms represent the percentage of cells in G1, S and G2-M phase. FACS profiles were analyzed using CellQuest software. Data are mean ± s.e.m. of four independent experiments. *P < 0.02 (t-test) (A). Representative images of a cell kinetic experiment. Asynchronous HeLa cells were pulse labeled with BrdU and released in BrdU-free medium. Cells were harvested and stained with anti-BrdU and propidium iodide (PI) at indicated time points (hours after release). R1 = early S-phase; R2 = mid-S-phase; R3 = late S/G2 phase (arrows indicate cells entered in new G1 phase) (B).
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DDU485F2: RNase H2 depletion causes a delay in S-G2 phases. Cell cycle distribution of asynchronous HeLa cells. Histograms represent the percentage of cells in G1, S and G2-M phase. FACS profiles were analyzed using CellQuest software. Data are mean ± s.e.m. of four independent experiments. *P < 0.02 (t-test) (A). Representative images of a cell kinetic experiment. Asynchronous HeLa cells were pulse labeled with BrdU and released in BrdU-free medium. Cells were harvested and stained with anti-BrdU and propidium iodide (PI) at indicated time points (hours after release). R1 = early S-phase; R2 = mid-S-phase; R3 = late S/G2 phase (arrows indicate cells entered in new G1 phase) (B).

Mentions: Exponentially growing HeLa cells were labeled with BrdU to mark replicating cells, and analyzed by cytofluorimetry for total DNA content and for active replication. RNase H2 silencing affected cell cycle progression, resulting in a reproducible and significant accumulation of cells in S and G2-M phases (Fig. 2A). Cell cycle progression was further analyzed in a kinetic experiment where cells traversing S-phase were labeled with a pulse of BrdU and followed for 8 h after release in BrdU-free medium. When compared with the SCRAMBLE control, cells depleted for RNase H2 were delayed in late-S/G2 phase and were very slow in getting back into G1 phase (Fig. 2B), indicative of replication problems. Similar results were obtained using MRC5VI cells, indicating that it is not a cell-specific effect (Supplementary Material, Fig. S4A).Figure 2.


Reduction of hRNase H2 activity in Aicardi-Goutières syndrome cells leads to replication stress and genome instability.

Pizzi S, Sertic S, Orcesi S, Cereda C, Bianchi M, Jackson AP, Lazzaro F, Plevani P, Muzi-Falconi M - Hum. Mol. Genet. (2014)

RNase H2 depletion causes a delay in S-G2 phases. Cell cycle distribution of asynchronous HeLa cells. Histograms represent the percentage of cells in G1, S and G2-M phase. FACS profiles were analyzed using CellQuest software. Data are mean ± s.e.m. of four independent experiments. *P < 0.02 (t-test) (A). Representative images of a cell kinetic experiment. Asynchronous HeLa cells were pulse labeled with BrdU and released in BrdU-free medium. Cells were harvested and stained with anti-BrdU and propidium iodide (PI) at indicated time points (hours after release). R1 = early S-phase; R2 = mid-S-phase; R3 = late S/G2 phase (arrows indicate cells entered in new G1 phase) (B).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4291245&req=5

DDU485F2: RNase H2 depletion causes a delay in S-G2 phases. Cell cycle distribution of asynchronous HeLa cells. Histograms represent the percentage of cells in G1, S and G2-M phase. FACS profiles were analyzed using CellQuest software. Data are mean ± s.e.m. of four independent experiments. *P < 0.02 (t-test) (A). Representative images of a cell kinetic experiment. Asynchronous HeLa cells were pulse labeled with BrdU and released in BrdU-free medium. Cells were harvested and stained with anti-BrdU and propidium iodide (PI) at indicated time points (hours after release). R1 = early S-phase; R2 = mid-S-phase; R3 = late S/G2 phase (arrows indicate cells entered in new G1 phase) (B).
Mentions: Exponentially growing HeLa cells were labeled with BrdU to mark replicating cells, and analyzed by cytofluorimetry for total DNA content and for active replication. RNase H2 silencing affected cell cycle progression, resulting in a reproducible and significant accumulation of cells in S and G2-M phases (Fig. 2A). Cell cycle progression was further analyzed in a kinetic experiment where cells traversing S-phase were labeled with a pulse of BrdU and followed for 8 h after release in BrdU-free medium. When compared with the SCRAMBLE control, cells depleted for RNase H2 were delayed in late-S/G2 phase and were very slow in getting back into G1 phase (Fig. 2B), indicative of replication problems. Similar results were obtained using MRC5VI cells, indicating that it is not a cell-specific effect (Supplementary Material, Fig. S4A).Figure 2.

Bottom Line: Our data indicate that in human cells RNase H2 plays a crucial role in correcting rNMPs misincorporation, preventing DNA damage.Such protective function is compromised in AGS patients and may be linked to unscheduled immune responses.These findings may be relevant to shed further light on the mechanisms involved in AGS pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.

Show MeSH
Related in: MedlinePlus