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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 deficiency does not trigger replication fork asymmetryHCT116 cells were sequentially labeled with IdU (green) and CldU (red) and then subjected to single fiber replication analyses as described in the legend to Figure 1. For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (i.e., outside the red lines in B and C), the fork was deemed asymmetric. (A) Examples of symmetric and asymmetric forks. (B,C) The percentage of asymmetric forks was calculated for both Mus81-proficient (B) and Mus81-deficient (C) cells. For Mus81-proficient and Mus81-deficient cells, 55 and 85 forks were counted, respectively.
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Figure 2: Mus81 deficiency does not trigger replication fork asymmetryHCT116 cells were sequentially labeled with IdU (green) and CldU (red) and then subjected to single fiber replication analyses as described in the legend to Figure 1. For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (i.e., outside the red lines in B and C), the fork was deemed asymmetric. (A) Examples of symmetric and asymmetric forks. (B,C) The percentage of asymmetric forks was calculated for both Mus81-proficient (B) and Mus81-deficient (C) cells. For Mus81-proficient and Mus81-deficient cells, 55 and 85 forks were counted, respectively.

Mentions: Shorter replication tracks reflect either a slower rate of DNA synthesis or replication fork stalling, preventing DNA synthesis from proceeding during a significant length of time relative to the labeling period. To distinguish between these two possibilities, replication fork asymmetry was analyzed in both Mus81-deficient and Mus81-proficient cells. Using the sequential-labeling technique, a stalled replication fork typically results in an asymmetric replication track (i.e., tracks that emanate from a common origin but are significantly different in length) (Figure 2A). A single-origin replication signal in which one side was 33% longer than the other side was defined as an asymmetric track. (For representative examples, see the rightmost fork in the fiber shown in Figure 1B and the leftmost fork in the fiber shown in Supplemental Figure 2C.) The frequency of asymmetric replication tracks was similar in both Mus81-deficient and proficient cells (Figure 2B and 2C) suggesting that Mus81 deficiency did not cause replication to stall. These observations suggest that the shorter replication tracks observed in Mus81-deficient cells result from slower DNA synthesis rates in those cells.


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 deficiency does not trigger replication fork asymmetryHCT116 cells were sequentially labeled with IdU (green) and CldU (red) and then subjected to single fiber replication analyses as described in the legend to Figure 1. For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (i.e., outside the red lines in B and C), the fork was deemed asymmetric. (A) Examples of symmetric and asymmetric forks. (B,C) The percentage of asymmetric forks was calculated for both Mus81-proficient (B) and Mus81-deficient (C) cells. For Mus81-proficient and Mus81-deficient cells, 55 and 85 forks were counted, respectively.
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Figure 2: Mus81 deficiency does not trigger replication fork asymmetryHCT116 cells were sequentially labeled with IdU (green) and CldU (red) and then subjected to single fiber replication analyses as described in the legend to Figure 1. For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (i.e., outside the red lines in B and C), the fork was deemed asymmetric. (A) Examples of symmetric and asymmetric forks. (B,C) The percentage of asymmetric forks was calculated for both Mus81-proficient (B) and Mus81-deficient (C) cells. For Mus81-proficient and Mus81-deficient cells, 55 and 85 forks were counted, respectively.
Mentions: Shorter replication tracks reflect either a slower rate of DNA synthesis or replication fork stalling, preventing DNA synthesis from proceeding during a significant length of time relative to the labeling period. To distinguish between these two possibilities, replication fork asymmetry was analyzed in both Mus81-deficient and Mus81-proficient cells. Using the sequential-labeling technique, a stalled replication fork typically results in an asymmetric replication track (i.e., tracks that emanate from a common origin but are significantly different in length) (Figure 2A). A single-origin replication signal in which one side was 33% longer than the other side was defined as an asymmetric track. (For representative examples, see the rightmost fork in the fiber shown in Figure 1B and the leftmost fork in the fiber shown in Supplemental Figure 2C.) The frequency of asymmetric replication tracks was similar in both Mus81-deficient and proficient cells (Figure 2B and 2C) suggesting that Mus81 deficiency did not cause replication to stall. These observations suggest that the shorter replication tracks observed in Mus81-deficient cells result from slower DNA synthesis rates in those cells.

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