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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

Mus81 endonuclease activity prevents irreversible fork stalling in response to perturbed replication(A) Cells were labeled with IdU (green) for 10 min, then CldU (red) for 20 min. A low dose of APH was included during the CldU incubation period (left). Cells were harvested for single DNA fiber spread analysis. The right schematic graph showed 3 typical replication tracks: regular fork, slow fork and stalled fork based on the length of red replication tracks affected by APH treatment. (B) Images of DNA fibers from Mus81-proficient (HCT116) and Mus81–deficient (Mus81−/−) cells. Five representative fibers for each image were extracted and aligned to the right of their respective images. Effects of APH are shown in the bottom panels.Fibers that replicated with a similar pace during the IdU (green) and CldU (red) labeling periods exhibited a red signal that was longer than the green signal (regular fork), and most of replication forks in the control samples for both HCT116 and Mus81−/− cells belong to this category (2 top panels); fibers that slowed replication during the CldU labeling period exhibited a red signal that was clearly shorter than the green signal (slow fork), and most of replication forks in APH treated HCT116 cells belong to this category (bottom left panel); and fibers that could not recover from replication inhibition exhibited green signals and no red signals (stalled), and most of replication forks in APH treated Mus81−/− cells belong to this category (bottom right panel). (C) Images of DNA fibers from Mus81-deficient cells that express wild type Mus81 (Mus81−/− com-wt Mus81) or mutant Mus81 (Mus81−/−comp-mutt Mus81). Mutant Mus81 lacks endonuclease activity. Effects of APH are shown in the bottom panels. Five representative fibers from APH treated wild-type Mus81 and mutant Mus81 samples (figure 7C, 2 bottom panels) were selected and aligned under their original images, respectively. Most of wild-type Mus81 cells showed slow forks while most of mutant-type Mus81 cells showed stalled forks after low dose of APH treatment.
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Figure 7: Mus81 endonuclease activity prevents irreversible fork stalling in response to perturbed replication(A) Cells were labeled with IdU (green) for 10 min, then CldU (red) for 20 min. A low dose of APH was included during the CldU incubation period (left). Cells were harvested for single DNA fiber spread analysis. The right schematic graph showed 3 typical replication tracks: regular fork, slow fork and stalled fork based on the length of red replication tracks affected by APH treatment. (B) Images of DNA fibers from Mus81-proficient (HCT116) and Mus81–deficient (Mus81−/−) cells. Five representative fibers for each image were extracted and aligned to the right of their respective images. Effects of APH are shown in the bottom panels.Fibers that replicated with a similar pace during the IdU (green) and CldU (red) labeling periods exhibited a red signal that was longer than the green signal (regular fork), and most of replication forks in the control samples for both HCT116 and Mus81−/− cells belong to this category (2 top panels); fibers that slowed replication during the CldU labeling period exhibited a red signal that was clearly shorter than the green signal (slow fork), and most of replication forks in APH treated HCT116 cells belong to this category (bottom left panel); and fibers that could not recover from replication inhibition exhibited green signals and no red signals (stalled), and most of replication forks in APH treated Mus81−/− cells belong to this category (bottom right panel). (C) Images of DNA fibers from Mus81-deficient cells that express wild type Mus81 (Mus81−/− com-wt Mus81) or mutant Mus81 (Mus81−/−comp-mutt Mus81). Mutant Mus81 lacks endonuclease activity. Effects of APH are shown in the bottom panels. Five representative fibers from APH treated wild-type Mus81 and mutant Mus81 samples (figure 7C, 2 bottom panels) were selected and aligned under their original images, respectively. Most of wild-type Mus81 cells showed slow forks while most of mutant-type Mus81 cells showed stalled forks after low dose of APH treatment.

Mentions: Although no significant DNA double-strand breaks were detected in Mus81-deficient cells, colony formation assays demonstrated that Mus81-deficient cells were markedly more sensitive to aphidicolin than Mus81-proficient cells (Figure 6C). These results suggested that the transient breaks generated by Mus81 in cells exposed to replication stress promoted cell survival by facilitating perturbed replication forks recovery. We asked, therefore, whether the endonuclease activity of Mus81 was involved in this process. Cells were labeled with IdU (green) for 10 min, then CldU (red) for 20 min. A low dose of APH was included during the CldU incubation period (Figure 7A). Cells were then harvested for single DNA fiber spread analysis 29, 30. During APH treatment, Mus81-proficient cells continued to replicate at a slow pace, whereas Mus81-deficient cells fully arrested replication (Figure 7B). This result is consistent with previous observations 30. Expression of wild-type Mus81 in Mus81-deficient cells rescued this phenotype, as a slow rate of DNA synthesis was observed during APH exposure (Figure 7C, left panels). When the endonuclease-dead version of Mus81 was expressed in Mus81-deficient cells, however, replication was not restored during APH exposure, as virtually no CldU incorporation was detected (Figure 7C, right panels). These results demonstrate that the endonuclease activity of Mus81, in addition to regulating the pace of DNA replication, facilitates the deceleration of DNA replication and prevents irreversible inhibition of DNA replication after exposure to low doses of APH.


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)

Mus81 endonuclease activity prevents irreversible fork stalling in response to perturbed replication(A) Cells were labeled with IdU (green) for 10 min, then CldU (red) for 20 min. A low dose of APH was included during the CldU incubation period (left). Cells were harvested for single DNA fiber spread analysis. The right schematic graph showed 3 typical replication tracks: regular fork, slow fork and stalled fork based on the length of red replication tracks affected by APH treatment. (B) Images of DNA fibers from Mus81-proficient (HCT116) and Mus81–deficient (Mus81−/−) cells. Five representative fibers for each image were extracted and aligned to the right of their respective images. Effects of APH are shown in the bottom panels.Fibers that replicated with a similar pace during the IdU (green) and CldU (red) labeling periods exhibited a red signal that was longer than the green signal (regular fork), and most of replication forks in the control samples for both HCT116 and Mus81−/− cells belong to this category (2 top panels); fibers that slowed replication during the CldU labeling period exhibited a red signal that was clearly shorter than the green signal (slow fork), and most of replication forks in APH treated HCT116 cells belong to this category (bottom left panel); and fibers that could not recover from replication inhibition exhibited green signals and no red signals (stalled), and most of replication forks in APH treated Mus81−/− cells belong to this category (bottom right panel). (C) Images of DNA fibers from Mus81-deficient cells that express wild type Mus81 (Mus81−/− com-wt Mus81) or mutant Mus81 (Mus81−/−comp-mutt Mus81). Mutant Mus81 lacks endonuclease activity. Effects of APH are shown in the bottom panels. Five representative fibers from APH treated wild-type Mus81 and mutant Mus81 samples (figure 7C, 2 bottom panels) were selected and aligned under their original images, respectively. Most of wild-type Mus81 cells showed slow forks while most of mutant-type Mus81 cells showed stalled forks after low dose of APH treatment.
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Figure 7: Mus81 endonuclease activity prevents irreversible fork stalling in response to perturbed replication(A) Cells were labeled with IdU (green) for 10 min, then CldU (red) for 20 min. A low dose of APH was included during the CldU incubation period (left). Cells were harvested for single DNA fiber spread analysis. The right schematic graph showed 3 typical replication tracks: regular fork, slow fork and stalled fork based on the length of red replication tracks affected by APH treatment. (B) Images of DNA fibers from Mus81-proficient (HCT116) and Mus81–deficient (Mus81−/−) cells. Five representative fibers for each image were extracted and aligned to the right of their respective images. Effects of APH are shown in the bottom panels.Fibers that replicated with a similar pace during the IdU (green) and CldU (red) labeling periods exhibited a red signal that was longer than the green signal (regular fork), and most of replication forks in the control samples for both HCT116 and Mus81−/− cells belong to this category (2 top panels); fibers that slowed replication during the CldU labeling period exhibited a red signal that was clearly shorter than the green signal (slow fork), and most of replication forks in APH treated HCT116 cells belong to this category (bottom left panel); and fibers that could not recover from replication inhibition exhibited green signals and no red signals (stalled), and most of replication forks in APH treated Mus81−/− cells belong to this category (bottom right panel). (C) Images of DNA fibers from Mus81-deficient cells that express wild type Mus81 (Mus81−/− com-wt Mus81) or mutant Mus81 (Mus81−/−comp-mutt Mus81). Mutant Mus81 lacks endonuclease activity. Effects of APH are shown in the bottom panels. Five representative fibers from APH treated wild-type Mus81 and mutant Mus81 samples (figure 7C, 2 bottom panels) were selected and aligned under their original images, respectively. Most of wild-type Mus81 cells showed slow forks while most of mutant-type Mus81 cells showed stalled forks after low dose of APH treatment.
Mentions: Although no significant DNA double-strand breaks were detected in Mus81-deficient cells, colony formation assays demonstrated that Mus81-deficient cells were markedly more sensitive to aphidicolin than Mus81-proficient cells (Figure 6C). These results suggested that the transient breaks generated by Mus81 in cells exposed to replication stress promoted cell survival by facilitating perturbed replication forks recovery. We asked, therefore, whether the endonuclease activity of Mus81 was involved in this process. Cells were labeled with IdU (green) for 10 min, then CldU (red) for 20 min. A low dose of APH was included during the CldU incubation period (Figure 7A). Cells were then harvested for single DNA fiber spread analysis 29, 30. During APH treatment, Mus81-proficient cells continued to replicate at a slow pace, whereas Mus81-deficient cells fully arrested replication (Figure 7B). This result is consistent with previous observations 30. Expression of wild-type Mus81 in Mus81-deficient cells rescued this phenotype, as a slow rate of DNA synthesis was observed during APH exposure (Figure 7C, left panels). When the endonuclease-dead version of Mus81 was expressed in Mus81-deficient cells, however, replication was not restored during APH exposure, as virtually no CldU incorporation was detected (Figure 7C, right panels). These results demonstrate that the endonuclease activity of Mus81, in addition to regulating the pace of DNA replication, facilitates the deceleration of DNA replication and prevents irreversible inhibition of DNA replication after exposure to low doses of APH.

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