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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 replication fork progression in Mus81−/− cellsHCT116 and Mus81−/− cells were transfected with siRNA directed against XPF or control siRNA. (A) Western-blot analysis revealed an efficient knockdown of XPF protein via siRNA treatment. (B-C) Cells were sequentially pulsed with IdU and CldU for 20 minutes, respectively before harvesting for DNA combing. Scatter plot was used to show the distribution of replication fork speed (B) and interorigin distance (C). Median with interquartile range was shown for the scatter plot. Mann Whitney test was used for the statistical analysis. A few extreme numbers (>4.5 kb/min for fork speed, >400 kb for origin distance) were not shown for a better resolution of the scatter plot. Statistical analyses were shown in Supplementary Table 4.
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Figure 8: Effects of depletion of XPF on replication fork progression in Mus81−/− cellsHCT116 and Mus81−/− cells were transfected with siRNA directed against XPF or control siRNA. (A) Western-blot analysis revealed an efficient knockdown of XPF protein via siRNA treatment. (B-C) Cells were sequentially pulsed with IdU and CldU for 20 minutes, respectively before harvesting for DNA combing. Scatter plot was used to show the distribution of replication fork speed (B) and interorigin distance (C). Median with interquartile range was shown for the scatter plot. Mann Whitney test was used for the statistical analysis. A few extreme numbers (>4.5 kb/min for fork speed, >400 kb for origin distance) were not shown for a better resolution of the scatter plot. Statistical analyses were shown in Supplementary Table 4.

Mentions: Our observations suggest that the endonuclease activity of Mus81 regulates both rates of DNA replication and recovery from mild DNA-replication perturbations. However, Mus81 did not affect cell survival during unperturbed growth. XPF is another member of the Mus81/XPF family, which plays important roles in the repair of DNA lesions beyond nucleotide excision repair 44. We asked, therefore, whether XPF could compensate for Mus81 deficiency during unperturbed growth. Mus81-proficient and Mus81-deficient HCT116 cells were transfected with siRNA directed against XPF (two times within 72 h). XPF expression was depleted 3 days after the second transfection (Figure 8A). As shown in Figure 8B, XPF depletion decreased replication fork speed in both Mus81 wild type and Mus81-deficient HCT116 cells. Notably, however, XPF depletion by itself affected DNA replication rates to a lesser extent than Mus81 depletion, and double depletion of Mus81 and XPF did not exhibit synergistic effects. Similar changes in inter-origin distances were shown in Figure 8C.


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 replication fork progression in Mus81−/− cellsHCT116 and Mus81−/− cells were transfected with siRNA directed against XPF or control siRNA. (A) Western-blot analysis revealed an efficient knockdown of XPF protein via siRNA treatment. (B-C) Cells were sequentially pulsed with IdU and CldU for 20 minutes, respectively before harvesting for DNA combing. Scatter plot was used to show the distribution of replication fork speed (B) and interorigin distance (C). Median with interquartile range was shown for the scatter plot. Mann Whitney test was used for the statistical analysis. A few extreme numbers (>4.5 kb/min for fork speed, >400 kb for origin distance) were not shown for a better resolution of the scatter plot. Statistical analyses were shown in Supplementary Table 4.
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Related In: Results  -  Collection

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Figure 8: Effects of depletion of XPF on replication fork progression in Mus81−/− cellsHCT116 and Mus81−/− cells were transfected with siRNA directed against XPF or control siRNA. (A) Western-blot analysis revealed an efficient knockdown of XPF protein via siRNA treatment. (B-C) Cells were sequentially pulsed with IdU and CldU for 20 minutes, respectively before harvesting for DNA combing. Scatter plot was used to show the distribution of replication fork speed (B) and interorigin distance (C). Median with interquartile range was shown for the scatter plot. Mann Whitney test was used for the statistical analysis. A few extreme numbers (>4.5 kb/min for fork speed, >400 kb for origin distance) were not shown for a better resolution of the scatter plot. Statistical analyses were shown in Supplementary Table 4.
Mentions: Our observations suggest that the endonuclease activity of Mus81 regulates both rates of DNA replication and recovery from mild DNA-replication perturbations. However, Mus81 did not affect cell survival during unperturbed growth. XPF is another member of the Mus81/XPF family, which plays important roles in the repair of DNA lesions beyond nucleotide excision repair 44. We asked, therefore, whether XPF could compensate for Mus81 deficiency during unperturbed growth. Mus81-proficient and Mus81-deficient HCT116 cells were transfected with siRNA directed against XPF (two times within 72 h). XPF expression was depleted 3 days after the second transfection (Figure 8A). As shown in Figure 8B, XPF depletion decreased replication fork speed in both Mus81 wild type and Mus81-deficient HCT116 cells. Notably, however, XPF depletion by itself affected DNA replication rates to a lesser extent than Mus81 depletion, and double depletion of Mus81 and XPF did not exhibit synergistic effects. Similar changes in inter-origin distances were shown in Figure 8C.

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