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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 depletion does not affect replication initiation patternsLocations of replication initiation events were determined by massively parallel sequencing of nascent strands from Mus81-proficient HCT116 cells and from HCT116 cells that lacked Mus81 (designated Mus81 (−/−). (A–C) Initiation profiles were visualized using the Broad Institute Integrated Genome Browser. Representative screen shots of replication-initiation profiles from three genomic loci are shown at different zoom levels. These loci include (A) 72 kb from the hemoglobin, beta (HBB) locus on Chromosome 11, (B) 195 kb from the CCCTC-binding factor (CTCF) locus on Chromosome 16, and (C) 1857 kb from Chromosome 14. It should be noted that the genome browser images are presented on a relative scale; therefore, the sizes of the peaks (calibrated per total numbers of aligned reads in each sample) indicate that initiation events preferentially occur at particular genomic locations but do not directly correspond to absolute initiation frequencies. (D) Locations of replication initiation events were determined by massively parallel sequencing of nascent strands from: 1) HCT116 cells (two separately sequenced samples designated HCT116 (1) and HCT116 (2)), 2) HCT116 cells that lacked Mus81 (designated Mus81 (−/−)), and 3) MCF7 cells. Locations of replication initiation sites were calculated using two bin sizes: 100 and 1000 bp. Shown are Pearson’s correlation coefficients concerning the location of replication initiation sites for comparisons between the HCT116 (1) sample and indicated samples.
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Figure 4: Mus81 depletion does not affect replication initiation patternsLocations of replication initiation events were determined by massively parallel sequencing of nascent strands from Mus81-proficient HCT116 cells and from HCT116 cells that lacked Mus81 (designated Mus81 (−/−). (A–C) Initiation profiles were visualized using the Broad Institute Integrated Genome Browser. Representative screen shots of replication-initiation profiles from three genomic loci are shown at different zoom levels. These loci include (A) 72 kb from the hemoglobin, beta (HBB) locus on Chromosome 11, (B) 195 kb from the CCCTC-binding factor (CTCF) locus on Chromosome 16, and (C) 1857 kb from Chromosome 14. It should be noted that the genome browser images are presented on a relative scale; therefore, the sizes of the peaks (calibrated per total numbers of aligned reads in each sample) indicate that initiation events preferentially occur at particular genomic locations but do not directly correspond to absolute initiation frequencies. (D) Locations of replication initiation events were determined by massively parallel sequencing of nascent strands from: 1) HCT116 cells (two separately sequenced samples designated HCT116 (1) and HCT116 (2)), 2) HCT116 cells that lacked Mus81 (designated Mus81 (−/−)), and 3) MCF7 cells. Locations of replication initiation sites were calculated using two bin sizes: 100 and 1000 bp. Shown are Pearson’s correlation coefficients concerning the location of replication initiation sites for comparisons between the HCT116 (1) sample and indicated samples.

Mentions: Slow replication can activate latent origins that do not initiate replication in rapidly replicating chromosomal loci 13. We asked, therefore, whether the increase in initiation frequency that was associated with Mus81 loss-of-function also reflected the activation of latent, cryptic, unused origins. Using both Mus81-proficient and Mus81-deficient cell lines we mapped all replication initiation events, as described 34. Massively parallel sequencing of newly replicated nascent DNA strands revealed strikingly similar replication-origin usage between these two cell lines (Figure 4). Similar patterns were evident in images of replication initiation patterns in individual loci (For examples, see Figure 4A-C), suggesting that similar genomic regions preferentially initiate DNA replication in both Mus81-proficient and deficient cells. When the overall reads per kilobase per million aligned reads (RPKM) values were compared between the Mus81-proficient and deficient HCT116 cells (bins that contained zero reads in both samples were excluded), Pearson correlation coefficients were 0.96 to 0.98 depending on bin size (Figure 4D). Control measurements comparing two technical replicates of sequence data obtained from HCT116 cells, resulted in a Pearson correlation coefficient of 0.98. Similar trends were observed when 100 bp bins were used, although Pearson correlation coefficients were generally lower (Figure 4D). Since the molecular combing data had demonstrated that Mus81-deficient cells exhibit shorter inter-origin distances than Mus81-proficient cells, the similar initiation patterns exhibited by the Mus81-proficient and deficient cells implies that replication initiation events in Mus81-deficient cells occur more frequently from the same sequences used by Mus81-proficient cells. Therefore, the higher percentage of origins that are activated in Mus81-deficient cells resulted from increased utilization of existing, pre-licensed flexible origins. Although the frequency of initiation was higher in Mus81-deficient cells, we have not observed activation of genomic regions that were not utilized as replication initiation sites in Mus81-proficient 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 depletion does not affect replication initiation patternsLocations of replication initiation events were determined by massively parallel sequencing of nascent strands from Mus81-proficient HCT116 cells and from HCT116 cells that lacked Mus81 (designated Mus81 (−/−). (A–C) Initiation profiles were visualized using the Broad Institute Integrated Genome Browser. Representative screen shots of replication-initiation profiles from three genomic loci are shown at different zoom levels. These loci include (A) 72 kb from the hemoglobin, beta (HBB) locus on Chromosome 11, (B) 195 kb from the CCCTC-binding factor (CTCF) locus on Chromosome 16, and (C) 1857 kb from Chromosome 14. It should be noted that the genome browser images are presented on a relative scale; therefore, the sizes of the peaks (calibrated per total numbers of aligned reads in each sample) indicate that initiation events preferentially occur at particular genomic locations but do not directly correspond to absolute initiation frequencies. (D) Locations of replication initiation events were determined by massively parallel sequencing of nascent strands from: 1) HCT116 cells (two separately sequenced samples designated HCT116 (1) and HCT116 (2)), 2) HCT116 cells that lacked Mus81 (designated Mus81 (−/−)), and 3) MCF7 cells. Locations of replication initiation sites were calculated using two bin sizes: 100 and 1000 bp. Shown are Pearson’s correlation coefficients concerning the location of replication initiation sites for comparisons between the HCT116 (1) sample and indicated samples.
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Figure 4: Mus81 depletion does not affect replication initiation patternsLocations of replication initiation events were determined by massively parallel sequencing of nascent strands from Mus81-proficient HCT116 cells and from HCT116 cells that lacked Mus81 (designated Mus81 (−/−). (A–C) Initiation profiles were visualized using the Broad Institute Integrated Genome Browser. Representative screen shots of replication-initiation profiles from three genomic loci are shown at different zoom levels. These loci include (A) 72 kb from the hemoglobin, beta (HBB) locus on Chromosome 11, (B) 195 kb from the CCCTC-binding factor (CTCF) locus on Chromosome 16, and (C) 1857 kb from Chromosome 14. It should be noted that the genome browser images are presented on a relative scale; therefore, the sizes of the peaks (calibrated per total numbers of aligned reads in each sample) indicate that initiation events preferentially occur at particular genomic locations but do not directly correspond to absolute initiation frequencies. (D) Locations of replication initiation events were determined by massively parallel sequencing of nascent strands from: 1) HCT116 cells (two separately sequenced samples designated HCT116 (1) and HCT116 (2)), 2) HCT116 cells that lacked Mus81 (designated Mus81 (−/−)), and 3) MCF7 cells. Locations of replication initiation sites were calculated using two bin sizes: 100 and 1000 bp. Shown are Pearson’s correlation coefficients concerning the location of replication initiation sites for comparisons between the HCT116 (1) sample and indicated samples.
Mentions: Slow replication can activate latent origins that do not initiate replication in rapidly replicating chromosomal loci 13. We asked, therefore, whether the increase in initiation frequency that was associated with Mus81 loss-of-function also reflected the activation of latent, cryptic, unused origins. Using both Mus81-proficient and Mus81-deficient cell lines we mapped all replication initiation events, as described 34. Massively parallel sequencing of newly replicated nascent DNA strands revealed strikingly similar replication-origin usage between these two cell lines (Figure 4). Similar patterns were evident in images of replication initiation patterns in individual loci (For examples, see Figure 4A-C), suggesting that similar genomic regions preferentially initiate DNA replication in both Mus81-proficient and deficient cells. When the overall reads per kilobase per million aligned reads (RPKM) values were compared between the Mus81-proficient and deficient HCT116 cells (bins that contained zero reads in both samples were excluded), Pearson correlation coefficients were 0.96 to 0.98 depending on bin size (Figure 4D). Control measurements comparing two technical replicates of sequence data obtained from HCT116 cells, resulted in a Pearson correlation coefficient of 0.98. Similar trends were observed when 100 bp bins were used, although Pearson correlation coefficients were generally lower (Figure 4D). Since the molecular combing data had demonstrated that Mus81-deficient cells exhibit shorter inter-origin distances than Mus81-proficient cells, the similar initiation patterns exhibited by the Mus81-proficient and deficient cells implies that replication initiation events in Mus81-deficient cells occur more frequently from the same sequences used by Mus81-proficient cells. Therefore, the higher percentage of origins that are activated in Mus81-deficient cells resulted from increased utilization of existing, pre-licensed flexible origins. Although the frequency of initiation was higher in Mus81-deficient cells, we have not observed activation of genomic regions that were not utilized as replication initiation sites in Mus81-proficient 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