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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 decreases transient DNA breakage and increases sensitivity to low doses of aphidicolinMus81 −/− (Mus81-deficient cells) and Mus81-comp (Mus81 −/− cells complemented with exogeneous Mus81 as described previously30, 31) were exposed to carrier (A) or to 0.5 μg/ml (about 1.5 μM) of aphidicolin (B) for 10 minutes and collected for neutral Comet assay. (C) HCT116 cells, Mus81−/− cells and Mus81−/−comp cells were treated with aphidicolin for 2 hours. The survival rate of cells was determined by clonogenic assay.
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Figure 6: Mus81 deficiency decreases transient DNA breakage and increases sensitivity to low doses of aphidicolinMus81 −/− (Mus81-deficient cells) and Mus81-comp (Mus81 −/− cells complemented with exogeneous Mus81 as described previously30, 31) were exposed to carrier (A) or to 0.5 μg/ml (about 1.5 μM) of aphidicolin (B) for 10 minutes and collected for neutral Comet assay. (C) HCT116 cells, Mus81−/− cells and Mus81−/−comp cells were treated with aphidicolin for 2 hours. The survival rate of cells was determined by clonogenic assay.

Mentions: The Mus81 endonuclease induces DNA breakage during the repair of DNA damage (e.g. interstrand crosslinks and topoisomerase cleavage complexes)33, 37. Mus81 converts perturbed replication forks into DNA breaks, thereby facilitating the rapid resumption of DNA replication. This avoids irreversible inhibition of DNA synthesis, DNA damage, and activation of cell-cycle checkpoints 30, 38. Mus81 also cleaves stalled replication forks that are generated by topoisomerase I (TOP1) cleavage complexes 33 and is involved in DNA breakage at common fragile sites during early mitosis 39, 40. We measured the frequency of DNA breaks in Mus81-proficient and deficient cells using a comet assay. As shown in Figure 6A, Mus81-deficient cells exhibited similar, although somewhat lower levels of spontaneous DNA breaks than Mus81-proficient cells. To investigate whether Mus81 endonuclease activity played a role in the recovery from perturbed replication during S-phase, we exposed cells to low doses of aphidicolin (0.5 μg/ml) shown previously to slow down replication without triggering DNA damage in Mus81-proficient cells30, 38. When cells were exposed to 0.5 μg/ml aphidicolin for 10 minutes, Mus81-proficient cells exhibited high levels of double-strand breaks (Figure 6B) but the frequency of double-strand breaks did not notably change in Mus81-deficient cells (Figure 6B).


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 decreases transient DNA breakage and increases sensitivity to low doses of aphidicolinMus81 −/− (Mus81-deficient cells) and Mus81-comp (Mus81 −/− cells complemented with exogeneous Mus81 as described previously30, 31) were exposed to carrier (A) or to 0.5 μg/ml (about 1.5 μM) of aphidicolin (B) for 10 minutes and collected for neutral Comet assay. (C) HCT116 cells, Mus81−/− cells and Mus81−/−comp cells were treated with aphidicolin for 2 hours. The survival rate of cells was determined by clonogenic assay.
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Figure 6: Mus81 deficiency decreases transient DNA breakage and increases sensitivity to low doses of aphidicolinMus81 −/− (Mus81-deficient cells) and Mus81-comp (Mus81 −/− cells complemented with exogeneous Mus81 as described previously30, 31) were exposed to carrier (A) or to 0.5 μg/ml (about 1.5 μM) of aphidicolin (B) for 10 minutes and collected for neutral Comet assay. (C) HCT116 cells, Mus81−/− cells and Mus81−/−comp cells were treated with aphidicolin for 2 hours. The survival rate of cells was determined by clonogenic assay.
Mentions: The Mus81 endonuclease induces DNA breakage during the repair of DNA damage (e.g. interstrand crosslinks and topoisomerase cleavage complexes)33, 37. Mus81 converts perturbed replication forks into DNA breaks, thereby facilitating the rapid resumption of DNA replication. This avoids irreversible inhibition of DNA synthesis, DNA damage, and activation of cell-cycle checkpoints 30, 38. Mus81 also cleaves stalled replication forks that are generated by topoisomerase I (TOP1) cleavage complexes 33 and is involved in DNA breakage at common fragile sites during early mitosis 39, 40. We measured the frequency of DNA breaks in Mus81-proficient and deficient cells using a comet assay. As shown in Figure 6A, Mus81-deficient cells exhibited similar, although somewhat lower levels of spontaneous DNA breaks than Mus81-proficient cells. To investigate whether Mus81 endonuclease activity played a role in the recovery from perturbed replication during S-phase, we exposed cells to low doses of aphidicolin (0.5 μg/ml) shown previously to slow down replication without triggering DNA damage in Mus81-proficient cells30, 38. When cells were exposed to 0.5 μg/ml aphidicolin for 10 minutes, Mus81-proficient cells exhibited high levels of double-strand breaks (Figure 6B) but the frequency of double-strand breaks did not notably change in Mus81-deficient cells (Figure 6B).

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