Limits...
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 is required to maintain replication rates(A) Site-directed mutagenesis was used to replace two aspartic-acid residues within the conserved Mus81 nuclease domain (338 and 339) with alanine residues. (B–E) Wild type and mutant versions of Mus81 tagged with tGFP in a doxycyclin-inducible vector were introduced into HCT116 Mus81−/− cells and stable cell lines were established. (B) Western-blot analysis showed reproducible Mus81 expression in response to doxycycline for 72 hours in these stable cell lines. (C,D) Single fiber replication analyses were used to measure rates of replication fork progression (C) and inter-origin distances (D) in these cells after 72 hours of Mus81-tGFP induction by doxycycline. Cells complemented with the wild type Mus81 exhibited slower replication fork progression compared to the rates of DNA synthesis in the parental Mus81-proficient HCT116 cells shown in Figure 1, reflecting either experimental variation or incomplete complementation by Mus81. Cells expressing Mus81 with a mutant nuclease domain exhibited slower replication fork progression and more frequent initiation than cells expressing the wild-type Mus81. Statistical analyses are shown in Supplementary Table 3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400873&req=5

Figure 5: Mus81 endonuclease activity is required to maintain replication rates(A) Site-directed mutagenesis was used to replace two aspartic-acid residues within the conserved Mus81 nuclease domain (338 and 339) with alanine residues. (B–E) Wild type and mutant versions of Mus81 tagged with tGFP in a doxycyclin-inducible vector were introduced into HCT116 Mus81−/− cells and stable cell lines were established. (B) Western-blot analysis showed reproducible Mus81 expression in response to doxycycline for 72 hours in these stable cell lines. (C,D) Single fiber replication analyses were used to measure rates of replication fork progression (C) and inter-origin distances (D) in these cells after 72 hours of Mus81-tGFP induction by doxycycline. Cells complemented with the wild type Mus81 exhibited slower replication fork progression compared to the rates of DNA synthesis in the parental Mus81-proficient HCT116 cells shown in Figure 1, reflecting either experimental variation or incomplete complementation by Mus81. Cells expressing Mus81 with a mutant nuclease domain exhibited slower replication fork progression and more frequent initiation than cells expressing the wild-type Mus81. Statistical analyses are shown in Supplementary Table 3.

Mentions: Mus81 is a structure-specific endonuclease, and its endonuclease activity is required for DNA-damage repair. Mus81 is part of a large protein complex that contains numerous enzymes involved in DNA repair 35. We asked, therefore, whether it is the enzymatic activity of Mus81 that affects the rate of DNA replication. Alternatively, Mus81 could play a structural role in this process by recruiting other proteins (such as the Mus81-containing “super complex”) to chromatin. The endonuclease activity of Mus81 requires two aspartic-acid residues at positions 338 and 339 within the conserved nuclease domain 36. Substituting these amino acids with alanine abolishes endonuclease activity. We used site-directed mutagenesis to reconstitute this mutation, generating an “endonuclease-dead” version of Mus81 (Figure 5A). We then transfected the Mus81-deficient HCT116 cells with constructs that encoded either a wild type or endonuclease-dead version of Mus81 (Figure 5B). Neither of these constructs affected cell cycle progression of Mus81-deficient cells (Supplementary Fig. 1D). Complementation with the wild-type Mus81 partially restored the slow replication observed in the Mus81 deficient cells. Expression of endonuclease-dead Mus81 resulted in slower replication fork progression and shorter inter-origin distances than expression of wild-type Mus81 (Figure 5C, 5D, and Figure Supplementary Table 3). This suggests that the endonuclease activity of Mus81 is involved in regulating the pace of DNA replication.


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 is required to maintain replication rates(A) Site-directed mutagenesis was used to replace two aspartic-acid residues within the conserved Mus81 nuclease domain (338 and 339) with alanine residues. (B–E) Wild type and mutant versions of Mus81 tagged with tGFP in a doxycyclin-inducible vector were introduced into HCT116 Mus81−/− cells and stable cell lines were established. (B) Western-blot analysis showed reproducible Mus81 expression in response to doxycycline for 72 hours in these stable cell lines. (C,D) Single fiber replication analyses were used to measure rates of replication fork progression (C) and inter-origin distances (D) in these cells after 72 hours of Mus81-tGFP induction by doxycycline. Cells complemented with the wild type Mus81 exhibited slower replication fork progression compared to the rates of DNA synthesis in the parental Mus81-proficient HCT116 cells shown in Figure 1, reflecting either experimental variation or incomplete complementation by Mus81. Cells expressing Mus81 with a mutant nuclease domain exhibited slower replication fork progression and more frequent initiation than cells expressing the wild-type Mus81. Statistical analyses are shown in Supplementary Table 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400873&req=5

Figure 5: Mus81 endonuclease activity is required to maintain replication rates(A) Site-directed mutagenesis was used to replace two aspartic-acid residues within the conserved Mus81 nuclease domain (338 and 339) with alanine residues. (B–E) Wild type and mutant versions of Mus81 tagged with tGFP in a doxycyclin-inducible vector were introduced into HCT116 Mus81−/− cells and stable cell lines were established. (B) Western-blot analysis showed reproducible Mus81 expression in response to doxycycline for 72 hours in these stable cell lines. (C,D) Single fiber replication analyses were used to measure rates of replication fork progression (C) and inter-origin distances (D) in these cells after 72 hours of Mus81-tGFP induction by doxycycline. Cells complemented with the wild type Mus81 exhibited slower replication fork progression compared to the rates of DNA synthesis in the parental Mus81-proficient HCT116 cells shown in Figure 1, reflecting either experimental variation or incomplete complementation by Mus81. Cells expressing Mus81 with a mutant nuclease domain exhibited slower replication fork progression and more frequent initiation than cells expressing the wild-type Mus81. Statistical analyses are shown in Supplementary Table 3.
Mentions: Mus81 is a structure-specific endonuclease, and its endonuclease activity is required for DNA-damage repair. Mus81 is part of a large protein complex that contains numerous enzymes involved in DNA repair 35. We asked, therefore, whether it is the enzymatic activity of Mus81 that affects the rate of DNA replication. Alternatively, Mus81 could play a structural role in this process by recruiting other proteins (such as the Mus81-containing “super complex”) to chromatin. The endonuclease activity of Mus81 requires two aspartic-acid residues at positions 338 and 339 within the conserved nuclease domain 36. Substituting these amino acids with alanine abolishes endonuclease activity. We used site-directed mutagenesis to reconstitute this mutation, generating an “endonuclease-dead” version of Mus81 (Figure 5A). We then transfected the Mus81-deficient HCT116 cells with constructs that encoded either a wild type or endonuclease-dead version of Mus81 (Figure 5B). Neither of these constructs affected cell cycle progression of Mus81-deficient cells (Supplementary Fig. 1D). Complementation with the wild-type Mus81 partially restored the slow replication observed in the Mus81 deficient cells. Expression of endonuclease-dead Mus81 resulted in slower replication fork progression and shorter inter-origin distances than expression of wild-type Mus81 (Figure 5C, 5D, and Figure Supplementary Table 3). This suggests that the endonuclease activity of Mus81 is involved in regulating the pace of DNA replication.

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