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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

Acute Mus81 depletion triggers slower DNA synthesis and elevated initiation frequencyHCT116 cells were transfected withsiRNA directed against Mus81 or with control siRNA. (A) Western-blot analysis showed that HCT116 cells transfected with siRNA against Mus81 exhibited a dramatic knockdown of Mus81 protein, compared to cells transfected with control siRNA 48 and 72 hours after transfection. Cells transfected with Mus81 siRNA or control siRNA for 48 hours were used for single fiber replication analyses: rates of replication fork progression (B) and inter-origin distances (C) in siRNA-treated cells. In comparisons between control- and Mus81-siRNA treated cells, the Mann Whitney test revealed significant differences in both replication fork velocity and inter-origin distance (Supplementary Table 2).
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Figure 3: Acute Mus81 depletion triggers slower DNA synthesis and elevated initiation frequencyHCT116 cells were transfected withsiRNA directed against Mus81 or with control siRNA. (A) Western-blot analysis showed that HCT116 cells transfected with siRNA against Mus81 exhibited a dramatic knockdown of Mus81 protein, compared to cells transfected with control siRNA 48 and 72 hours after transfection. Cells transfected with Mus81 siRNA or control siRNA for 48 hours were used for single fiber replication analyses: rates of replication fork progression (B) and inter-origin distances (C) in siRNA-treated cells. In comparisons between control- and Mus81-siRNA treated cells, the Mann Whitney test revealed significant differences in both replication fork velocity and inter-origin distance (Supplementary Table 2).

Mentions: As the cell lines used for these analyses had been propagated in culture for several months, the Mus81 phenotype (i.e., the slow rate of DNA synthesis and the high rate of initiation frequency) may have resulted from long-term adaptation to the absence of Mus81. Alternatively, these phenotypes may indicate that Mus81 is constantly involved in determining the rates of replication fork progression and replication initiation. To distinguish between these possibilities, we asked what effect an acute knockdown of Mus81 would have on DNA replication. We used siRNA to deplete Mus8133 typically achieving complete depletion 2 days following the introduction of siRNA (Figure 3A). For cells exposed to siRNA directed against Mus81, we observed no significant cell-cycle changes (Supplementary Fig. 1C). When HCT116 cells were transfected with siRNA directed against Mus81, however, DNA synthesis was significantly slower compared to cells treated with control siRNA (Figure 3B and Supplementary Table 2). This suggests that both acute and chronic depletion of Mus81 results in slower replication fork progression. In addition, cells transfected with Mus81-specific siRNA exhibited shorter inter-origin distances, indicating an increased frequency of replication initiation events (Figure 3C and Supplementary Table 2). Similar results were obtained when the breast cancer cells MDA-MB-231 were used (Supplementary Fig. 3). These results indicate that the depletion of Mus81 rapidly slows DNA synthesis and increases the frequency of replication initiation events.


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)

Acute Mus81 depletion triggers slower DNA synthesis and elevated initiation frequencyHCT116 cells were transfected withsiRNA directed against Mus81 or with control siRNA. (A) Western-blot analysis showed that HCT116 cells transfected with siRNA against Mus81 exhibited a dramatic knockdown of Mus81 protein, compared to cells transfected with control siRNA 48 and 72 hours after transfection. Cells transfected with Mus81 siRNA or control siRNA for 48 hours were used for single fiber replication analyses: rates of replication fork progression (B) and inter-origin distances (C) in siRNA-treated cells. In comparisons between control- and Mus81-siRNA treated cells, the Mann Whitney test revealed significant differences in both replication fork velocity and inter-origin distance (Supplementary Table 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400873&req=5

Figure 3: Acute Mus81 depletion triggers slower DNA synthesis and elevated initiation frequencyHCT116 cells were transfected withsiRNA directed against Mus81 or with control siRNA. (A) Western-blot analysis showed that HCT116 cells transfected with siRNA against Mus81 exhibited a dramatic knockdown of Mus81 protein, compared to cells transfected with control siRNA 48 and 72 hours after transfection. Cells transfected with Mus81 siRNA or control siRNA for 48 hours were used for single fiber replication analyses: rates of replication fork progression (B) and inter-origin distances (C) in siRNA-treated cells. In comparisons between control- and Mus81-siRNA treated cells, the Mann Whitney test revealed significant differences in both replication fork velocity and inter-origin distance (Supplementary Table 2).
Mentions: As the cell lines used for these analyses had been propagated in culture for several months, the Mus81 phenotype (i.e., the slow rate of DNA synthesis and the high rate of initiation frequency) may have resulted from long-term adaptation to the absence of Mus81. Alternatively, these phenotypes may indicate that Mus81 is constantly involved in determining the rates of replication fork progression and replication initiation. To distinguish between these possibilities, we asked what effect an acute knockdown of Mus81 would have on DNA replication. We used siRNA to deplete Mus8133 typically achieving complete depletion 2 days following the introduction of siRNA (Figure 3A). For cells exposed to siRNA directed against Mus81, we observed no significant cell-cycle changes (Supplementary Fig. 1C). When HCT116 cells were transfected with siRNA directed against Mus81, however, DNA synthesis was significantly slower compared to cells treated with control siRNA (Figure 3B and Supplementary Table 2). This suggests that both acute and chronic depletion of Mus81 results in slower replication fork progression. In addition, cells transfected with Mus81-specific siRNA exhibited shorter inter-origin distances, indicating an increased frequency of replication initiation events (Figure 3C and Supplementary Table 2). Similar results were obtained when the breast cancer cells MDA-MB-231 were used (Supplementary Fig. 3). These results indicate that the depletion of Mus81 rapidly slows DNA synthesis and increases the frequency of replication initiation events.

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