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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

A high frequency of replication initiation and slower replication forks in Mus81 deficient cellsCells were sequentially labeled with ldU and CIdU. Extracted DNA was then stretched on silanized microscope coverslips. Regions of DNA that contained ldU and CIdU were fluorescently labeled via immunochemistry. DNA fibers were counterstained using an antibody directed against single-stranded DNA to exclude end-fiber replication tracks from progression-rate calculations, and to ensure that origins used to calculate an inter-origin distance were on the same fiber. (A) Raw images that include a FISH signal (top, traced by a yellow line) and a merged image of FISH (blue) and replication signals (green for IdU and red for CldU).A BAC of known size was used as the FISH probe for fiber size calibration. Scale bar, 50kb. (B) Typical replication signals (replicating DNA, top; anti-single strand DNA (ssDNA) antibody to trace DNA fiber, middle; Merged image, blue for ssDNA, bottom). The corresponding schematic diagram is shown underneath the fluorescence images (see Supplementary Fig. 2 for more DNA fiber examples). Replication initiation events occurred either during the initial IdU labeling (Ori1), during the secondary CldU labeling (Ori2), or before labeling was initiated (Ori3). Inter-origin distances were measured in fibers that contained at least two initiation events. Replication tracks that represented 20 min of DNA synthesis (a, c, e, f, g and h) were used to calculate replication fork velocity. Replication tracks that represented less than 20 min (b, d, initiated during a labeling), or that localized to the end of the DNA fiber (not showed) were not used. Scale bar, 20kb. (C, D). Data were binned and the percentage of replication signals that corresponded to a particular range of replication-rate (C) or inter-origin distance (D) was plotted for both Mus81-proficient (HCT116) and Mus81-deficient (Mus81−/−) cells. Mus81-deficient cells exhibited slower replication fork progression (C), and a higher frequency of replication initiation events (D). (C) and (D) are representative graphs from 3 experiments. Statistical analyses are shown in Supplementary Table 1.
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Figure 1: A high frequency of replication initiation and slower replication forks in Mus81 deficient cellsCells were sequentially labeled with ldU and CIdU. Extracted DNA was then stretched on silanized microscope coverslips. Regions of DNA that contained ldU and CIdU were fluorescently labeled via immunochemistry. DNA fibers were counterstained using an antibody directed against single-stranded DNA to exclude end-fiber replication tracks from progression-rate calculations, and to ensure that origins used to calculate an inter-origin distance were on the same fiber. (A) Raw images that include a FISH signal (top, traced by a yellow line) and a merged image of FISH (blue) and replication signals (green for IdU and red for CldU).A BAC of known size was used as the FISH probe for fiber size calibration. Scale bar, 50kb. (B) Typical replication signals (replicating DNA, top; anti-single strand DNA (ssDNA) antibody to trace DNA fiber, middle; Merged image, blue for ssDNA, bottom). The corresponding schematic diagram is shown underneath the fluorescence images (see Supplementary Fig. 2 for more DNA fiber examples). Replication initiation events occurred either during the initial IdU labeling (Ori1), during the secondary CldU labeling (Ori2), or before labeling was initiated (Ori3). Inter-origin distances were measured in fibers that contained at least two initiation events. Replication tracks that represented 20 min of DNA synthesis (a, c, e, f, g and h) were used to calculate replication fork velocity. Replication tracks that represented less than 20 min (b, d, initiated during a labeling), or that localized to the end of the DNA fiber (not showed) were not used. Scale bar, 20kb. (C, D). Data were binned and the percentage of replication signals that corresponded to a particular range of replication-rate (C) or inter-origin distance (D) was plotted for both Mus81-proficient (HCT116) and Mus81-deficient (Mus81−/−) cells. Mus81-deficient cells exhibited slower replication fork progression (C), and a higher frequency of replication initiation events (D). (C) and (D) are representative graphs from 3 experiments. Statistical analyses are shown in Supplementary Table 1.

Mentions: We measured rates of replication fork progression by sequentially labeling cells with the thymidine analogs iodo-deoxyuridine (ldU) and chloro-deoxyuridine (CIdU). DNA fibers were then obtained from labeled cells and stretched onto microscope slides. Regions of nucleotide-analog incorporation (replication tracks) were visualized on these stretched DNA fibers using immunofluorescence, as described 30, 32 (Figures 1A and 1B). Using this technique, most replication tracks appeared as regions of green fluorescence (IdU incorporation) adjacent to regions of red fluorescence (CldU incorporation). This represents the fluorescent signature of replication fork progression. Lengths associated with ldU and CIdU incorporation were measured and rates of replication fork progression were calculated from these values. Fluorescent in-situ hybridization (FISH) was used to confirm these size ranges. The inclusion of a FISH signal serves an internal DNA size control. Despite the inclusion of a FISH marker, accurate comparisons of replication fork progression rates using dynamic molecular combing require that the studied samples be labeled and processed at the same experiment because minor variations in the duration of the thymidine analog pulses can cause variations in the length of replication tracks. Comparisons among measurements obtained from separate biological replicates necessitate a common sample that can serve as a standard, as reported below.


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)

A high frequency of replication initiation and slower replication forks in Mus81 deficient cellsCells were sequentially labeled with ldU and CIdU. Extracted DNA was then stretched on silanized microscope coverslips. Regions of DNA that contained ldU and CIdU were fluorescently labeled via immunochemistry. DNA fibers were counterstained using an antibody directed against single-stranded DNA to exclude end-fiber replication tracks from progression-rate calculations, and to ensure that origins used to calculate an inter-origin distance were on the same fiber. (A) Raw images that include a FISH signal (top, traced by a yellow line) and a merged image of FISH (blue) and replication signals (green for IdU and red for CldU).A BAC of known size was used as the FISH probe for fiber size calibration. Scale bar, 50kb. (B) Typical replication signals (replicating DNA, top; anti-single strand DNA (ssDNA) antibody to trace DNA fiber, middle; Merged image, blue for ssDNA, bottom). The corresponding schematic diagram is shown underneath the fluorescence images (see Supplementary Fig. 2 for more DNA fiber examples). Replication initiation events occurred either during the initial IdU labeling (Ori1), during the secondary CldU labeling (Ori2), or before labeling was initiated (Ori3). Inter-origin distances were measured in fibers that contained at least two initiation events. Replication tracks that represented 20 min of DNA synthesis (a, c, e, f, g and h) were used to calculate replication fork velocity. Replication tracks that represented less than 20 min (b, d, initiated during a labeling), or that localized to the end of the DNA fiber (not showed) were not used. Scale bar, 20kb. (C, D). Data were binned and the percentage of replication signals that corresponded to a particular range of replication-rate (C) or inter-origin distance (D) was plotted for both Mus81-proficient (HCT116) and Mus81-deficient (Mus81−/−) cells. Mus81-deficient cells exhibited slower replication fork progression (C), and a higher frequency of replication initiation events (D). (C) and (D) are representative graphs from 3 experiments. Statistical analyses are shown in Supplementary Table 1.
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Related In: Results  -  Collection

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Figure 1: A high frequency of replication initiation and slower replication forks in Mus81 deficient cellsCells were sequentially labeled with ldU and CIdU. Extracted DNA was then stretched on silanized microscope coverslips. Regions of DNA that contained ldU and CIdU were fluorescently labeled via immunochemistry. DNA fibers were counterstained using an antibody directed against single-stranded DNA to exclude end-fiber replication tracks from progression-rate calculations, and to ensure that origins used to calculate an inter-origin distance were on the same fiber. (A) Raw images that include a FISH signal (top, traced by a yellow line) and a merged image of FISH (blue) and replication signals (green for IdU and red for CldU).A BAC of known size was used as the FISH probe for fiber size calibration. Scale bar, 50kb. (B) Typical replication signals (replicating DNA, top; anti-single strand DNA (ssDNA) antibody to trace DNA fiber, middle; Merged image, blue for ssDNA, bottom). The corresponding schematic diagram is shown underneath the fluorescence images (see Supplementary Fig. 2 for more DNA fiber examples). Replication initiation events occurred either during the initial IdU labeling (Ori1), during the secondary CldU labeling (Ori2), or before labeling was initiated (Ori3). Inter-origin distances were measured in fibers that contained at least two initiation events. Replication tracks that represented 20 min of DNA synthesis (a, c, e, f, g and h) were used to calculate replication fork velocity. Replication tracks that represented less than 20 min (b, d, initiated during a labeling), or that localized to the end of the DNA fiber (not showed) were not used. Scale bar, 20kb. (C, D). Data were binned and the percentage of replication signals that corresponded to a particular range of replication-rate (C) or inter-origin distance (D) was plotted for both Mus81-proficient (HCT116) and Mus81-deficient (Mus81−/−) cells. Mus81-deficient cells exhibited slower replication fork progression (C), and a higher frequency of replication initiation events (D). (C) and (D) are representative graphs from 3 experiments. Statistical analyses are shown in Supplementary Table 1.
Mentions: We measured rates of replication fork progression by sequentially labeling cells with the thymidine analogs iodo-deoxyuridine (ldU) and chloro-deoxyuridine (CIdU). DNA fibers were then obtained from labeled cells and stretched onto microscope slides. Regions of nucleotide-analog incorporation (replication tracks) were visualized on these stretched DNA fibers using immunofluorescence, as described 30, 32 (Figures 1A and 1B). Using this technique, most replication tracks appeared as regions of green fluorescence (IdU incorporation) adjacent to regions of red fluorescence (CldU incorporation). This represents the fluorescent signature of replication fork progression. Lengths associated with ldU and CIdU incorporation were measured and rates of replication fork progression were calculated from these values. Fluorescent in-situ hybridization (FISH) was used to confirm these size ranges. The inclusion of a FISH signal serves an internal DNA size control. Despite the inclusion of a FISH marker, accurate comparisons of replication fork progression rates using dynamic molecular combing require that the studied samples be labeled and processed at the same experiment because minor variations in the duration of the thymidine analog pulses can cause variations in the length of replication tracks. Comparisons among measurements obtained from separate biological replicates necessitate a common sample that can serve as a standard, as reported below.

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