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The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage.

Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim R, Shimura T, Pommier Y, Aladjem MI - Nat Commun (2015)

Bottom Line: Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells.Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth.These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.

No MeSH data available.


Related in: MedlinePlus

Effects of depletion of XPF on Mus81−/− Cell SurvivalHCT116 and Mus81−/− cells were transfected with control siRNA or XPF siRNA as described in Figure 8. (A) Cell cycle analysis by FACS. Cells were pulse-labeled with EdU for 45 min before harvesting. Cell cycle was tested with a Click-iT EdU kit by FACS. Examples of bivariate FACS were shown and percentage of cells in each cell cycle was shown in Supplementary Table 5. (B) Histogram of percentage of subG1 from Figure 9A. (C) The % of apoptotic cells was measured by TUNEL assay. Cells were transfected with control siRNA or XPF siRNA twice with 72 hours interval and collected 72 hours after the second transfection. TUNEL positive cells were analyzed by FACS (see Supplemental Figure 6B for the FACS dot plots). (D) Clonogenic assays. 24 hours after the second siRNA transfection, cells were seeded into 6-well plates at 50, 250, or 1000 cells per well. Two weeks later, cells were fixed and stained to count the colonies.
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Figure 9: Effects of depletion of XPF on Mus81−/− Cell SurvivalHCT116 and Mus81−/− cells were transfected with control siRNA or XPF siRNA as described in Figure 8. (A) Cell cycle analysis by FACS. Cells were pulse-labeled with EdU for 45 min before harvesting. Cell cycle was tested with a Click-iT EdU kit by FACS. Examples of bivariate FACS were shown and percentage of cells in each cell cycle was shown in Supplementary Table 5. (B) Histogram of percentage of subG1 from Figure 9A. (C) The % of apoptotic cells was measured by TUNEL assay. Cells were transfected with control siRNA or XPF siRNA twice with 72 hours interval and collected 72 hours after the second transfection. TUNEL positive cells were analyzed by FACS (see Supplemental Figure 6B for the FACS dot plots). (D) Clonogenic assays. 24 hours after the second siRNA transfection, cells were seeded into 6-well plates at 50, 250, or 1000 cells per well. Two weeks later, cells were fixed and stained to count the colonies.

Mentions: By contrast, when Mus81-deficient cells were treated with XPF siRNA, they exhibited a dramatic increase in sub-G1 or apoptotic cells as measured by flow cytometry analyses (Figure 9A, B and Supplementary Table 5). Annexin V and TUNEL apoptotic assay confirmed that there were more apoptotic cells in Mus81-deficient cells when XPF was depleted (Figure 9C, Supplementary Fig. 6). In agreement, clonogenic assays showed that depletion of XPF markedly decreased the survival of HCT116 cells. This effect was more pronounced in Mus81-deficient cells (Figure 9D). These results suggest that XPF may act in concert with Mus81 to prevent the formation of deleterious DNA lesions. These results also demonstrate that at least one of the two endonucleases is required for the normal progression of DNA synthesis during an unperturbed cell cycle, and that XPF may promote cell survival in the absence of Mus81 during unperturbed growth.


The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage.

Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim R, Shimura T, Pommier Y, Aladjem MI - Nat Commun (2015)

Effects of depletion of XPF on Mus81−/− Cell SurvivalHCT116 and Mus81−/− cells were transfected with control siRNA or XPF siRNA as described in Figure 8. (A) Cell cycle analysis by FACS. Cells were pulse-labeled with EdU for 45 min before harvesting. Cell cycle was tested with a Click-iT EdU kit by FACS. Examples of bivariate FACS were shown and percentage of cells in each cell cycle was shown in Supplementary Table 5. (B) Histogram of percentage of subG1 from Figure 9A. (C) The % of apoptotic cells was measured by TUNEL assay. Cells were transfected with control siRNA or XPF siRNA twice with 72 hours interval and collected 72 hours after the second transfection. TUNEL positive cells were analyzed by FACS (see Supplemental Figure 6B for the FACS dot plots). (D) Clonogenic assays. 24 hours after the second siRNA transfection, cells were seeded into 6-well plates at 50, 250, or 1000 cells per well. Two weeks later, cells were fixed and stained to count the colonies.
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Figure 9: Effects of depletion of XPF on Mus81−/− Cell SurvivalHCT116 and Mus81−/− cells were transfected with control siRNA or XPF siRNA as described in Figure 8. (A) Cell cycle analysis by FACS. Cells were pulse-labeled with EdU for 45 min before harvesting. Cell cycle was tested with a Click-iT EdU kit by FACS. Examples of bivariate FACS were shown and percentage of cells in each cell cycle was shown in Supplementary Table 5. (B) Histogram of percentage of subG1 from Figure 9A. (C) The % of apoptotic cells was measured by TUNEL assay. Cells were transfected with control siRNA or XPF siRNA twice with 72 hours interval and collected 72 hours after the second transfection. TUNEL positive cells were analyzed by FACS (see Supplemental Figure 6B for the FACS dot plots). (D) Clonogenic assays. 24 hours after the second siRNA transfection, cells were seeded into 6-well plates at 50, 250, or 1000 cells per well. Two weeks later, cells were fixed and stained to count the colonies.
Mentions: By contrast, when Mus81-deficient cells were treated with XPF siRNA, they exhibited a dramatic increase in sub-G1 or apoptotic cells as measured by flow cytometry analyses (Figure 9A, B and Supplementary Table 5). Annexin V and TUNEL apoptotic assay confirmed that there were more apoptotic cells in Mus81-deficient cells when XPF was depleted (Figure 9C, Supplementary Fig. 6). In agreement, clonogenic assays showed that depletion of XPF markedly decreased the survival of HCT116 cells. This effect was more pronounced in Mus81-deficient cells (Figure 9D). These results suggest that XPF may act in concert with Mus81 to prevent the formation of deleterious DNA lesions. These results also demonstrate that at least one of the two endonucleases is required for the normal progression of DNA synthesis during an unperturbed cell cycle, and that XPF may promote cell survival in the absence of Mus81 during unperturbed growth.

Bottom Line: Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells.Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth.These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.

No MeSH data available.


Related in: MedlinePlus