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Checkpoint independence of most DNA replication origins in fission yeast.

Mickle KL, Ramanathan S, Rosebrock A, Oliva A, Chaudari A, Yompakdee C, Scott D, Leatherwood J, Huberman JA - BMC Mol. Biol. (2007)

Bottom Line: Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories.We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU.The fact that approximately 97% of fission yeast replication origins - both early and late - are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, SUNY at Stony Brook, Stony Brook, New York 11794-5222, USA. katie.mickle@gmail.com

ABSTRACT

Background: In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results: Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion: The fact that approximately 97% of fission yeast replication origins - both early and late - are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

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Locations and efficiencies of putative origins on the three chromosomes. The chromosomes are shown as consecutive horizontal lines, 1 Mbp per line. The position of the centromere on each chromosome is indicated by a light yellow rectangle. The positions of origins classified as strong, medium, weak or very weak are identified by vertical lines. The lines range in color from red (strong) to brown (very weak) and from long (strong) to short (very weak). The positions of potential origins below the detection limit are indicated by the text character, "0", and ambiguous origins (where our probes were too widely spaced to permit confident evaluation) are shown by the character, "?". Small circles above each chromosome line indicate the positions of origins identified by Heichinger et al. ([14]; top row of circles; light blue), AT islands (next row of circles; magenta), origins identified by Feng et al. [34] in cds1Δ cells (next row; orange) or in wild-type cells (next row; purple), and pre-RCs identified by Hayashi et al. ([15]; bottom row; dark blue or red circles or squares). For the pre-RCs, the colors blue and red distinguish the pre-RCs that are late/weak or early/strong, respectively. The circles represent pre-RCs that are not affected by deletion of cds1, while the squares indicate pre-RCs that replicate to a greater extent in cds1Δ cells than in wild-type cells [15]. The positions of origins where the signals (our measurements; Additional Files 4, 5, 6) for both checkpoint-mutant strains were significantly greater than the signal for wild-type cells are indicated by the text character, "C", and the positions of origins with the opposite characteristic (wild-type signal significantly greater than the signals from both checkpoint-mutant strains) are shown by the text character, "W". A pale green background indicates a large region with a high frequency of stronger origins. A pale yellow background indicates a large region with a high frequency of weaker origins. (A) chromosome 1; (B) chromosome 2; (C) chromosome 3.
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Figure 6: Locations and efficiencies of putative origins on the three chromosomes. The chromosomes are shown as consecutive horizontal lines, 1 Mbp per line. The position of the centromere on each chromosome is indicated by a light yellow rectangle. The positions of origins classified as strong, medium, weak or very weak are identified by vertical lines. The lines range in color from red (strong) to brown (very weak) and from long (strong) to short (very weak). The positions of potential origins below the detection limit are indicated by the text character, "0", and ambiguous origins (where our probes were too widely spaced to permit confident evaluation) are shown by the character, "?". Small circles above each chromosome line indicate the positions of origins identified by Heichinger et al. ([14]; top row of circles; light blue), AT islands (next row of circles; magenta), origins identified by Feng et al. [34] in cds1Δ cells (next row; orange) or in wild-type cells (next row; purple), and pre-RCs identified by Hayashi et al. ([15]; bottom row; dark blue or red circles or squares). For the pre-RCs, the colors blue and red distinguish the pre-RCs that are late/weak or early/strong, respectively. The circles represent pre-RCs that are not affected by deletion of cds1, while the squares indicate pre-RCs that replicate to a greater extent in cds1Δ cells than in wild-type cells [15]. The positions of origins where the signals (our measurements; Additional Files 4, 5, 6) for both checkpoint-mutant strains were significantly greater than the signal for wild-type cells are indicated by the text character, "C", and the positions of origins with the opposite characteristic (wild-type signal significantly greater than the signals from both checkpoint-mutant strains) are shown by the text character, "W". A pale green background indicates a large region with a high frequency of stronger origins. A pale yellow background indicates a large region with a high frequency of weaker origins. (A) chromosome 1; (B) chromosome 2; (C) chromosome 3.

Mentions: On the basis of extent of incorporation of BrdU in the presence of HU, Hayashi et al. [15] divided the pre-RCs that they identified into two classes: strong/early (identified by red circles and squares in our Figs. 2 and 6) and weak/late (identified by dark blue circles and squares in our Figs. 2, 6 and 8). As shown in Fig. 3C (based on results in Additional File 7), these two classes corresponded well with our five classes. The origins that we classified as below limit or very weak were mostly weak/late according to Hayashi et al., while the origins that we classified as medium or strong were almost entirely strong/early by their classification [15].


Checkpoint independence of most DNA replication origins in fission yeast.

Mickle KL, Ramanathan S, Rosebrock A, Oliva A, Chaudari A, Yompakdee C, Scott D, Leatherwood J, Huberman JA - BMC Mol. Biol. (2007)

Locations and efficiencies of putative origins on the three chromosomes. The chromosomes are shown as consecutive horizontal lines, 1 Mbp per line. The position of the centromere on each chromosome is indicated by a light yellow rectangle. The positions of origins classified as strong, medium, weak or very weak are identified by vertical lines. The lines range in color from red (strong) to brown (very weak) and from long (strong) to short (very weak). The positions of potential origins below the detection limit are indicated by the text character, "0", and ambiguous origins (where our probes were too widely spaced to permit confident evaluation) are shown by the character, "?". Small circles above each chromosome line indicate the positions of origins identified by Heichinger et al. ([14]; top row of circles; light blue), AT islands (next row of circles; magenta), origins identified by Feng et al. [34] in cds1Δ cells (next row; orange) or in wild-type cells (next row; purple), and pre-RCs identified by Hayashi et al. ([15]; bottom row; dark blue or red circles or squares). For the pre-RCs, the colors blue and red distinguish the pre-RCs that are late/weak or early/strong, respectively. The circles represent pre-RCs that are not affected by deletion of cds1, while the squares indicate pre-RCs that replicate to a greater extent in cds1Δ cells than in wild-type cells [15]. The positions of origins where the signals (our measurements; Additional Files 4, 5, 6) for both checkpoint-mutant strains were significantly greater than the signal for wild-type cells are indicated by the text character, "C", and the positions of origins with the opposite characteristic (wild-type signal significantly greater than the signals from both checkpoint-mutant strains) are shown by the text character, "W". A pale green background indicates a large region with a high frequency of stronger origins. A pale yellow background indicates a large region with a high frequency of weaker origins. (A) chromosome 1; (B) chromosome 2; (C) chromosome 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Locations and efficiencies of putative origins on the three chromosomes. The chromosomes are shown as consecutive horizontal lines, 1 Mbp per line. The position of the centromere on each chromosome is indicated by a light yellow rectangle. The positions of origins classified as strong, medium, weak or very weak are identified by vertical lines. The lines range in color from red (strong) to brown (very weak) and from long (strong) to short (very weak). The positions of potential origins below the detection limit are indicated by the text character, "0", and ambiguous origins (where our probes were too widely spaced to permit confident evaluation) are shown by the character, "?". Small circles above each chromosome line indicate the positions of origins identified by Heichinger et al. ([14]; top row of circles; light blue), AT islands (next row of circles; magenta), origins identified by Feng et al. [34] in cds1Δ cells (next row; orange) or in wild-type cells (next row; purple), and pre-RCs identified by Hayashi et al. ([15]; bottom row; dark blue or red circles or squares). For the pre-RCs, the colors blue and red distinguish the pre-RCs that are late/weak or early/strong, respectively. The circles represent pre-RCs that are not affected by deletion of cds1, while the squares indicate pre-RCs that replicate to a greater extent in cds1Δ cells than in wild-type cells [15]. The positions of origins where the signals (our measurements; Additional Files 4, 5, 6) for both checkpoint-mutant strains were significantly greater than the signal for wild-type cells are indicated by the text character, "C", and the positions of origins with the opposite characteristic (wild-type signal significantly greater than the signals from both checkpoint-mutant strains) are shown by the text character, "W". A pale green background indicates a large region with a high frequency of stronger origins. A pale yellow background indicates a large region with a high frequency of weaker origins. (A) chromosome 1; (B) chromosome 2; (C) chromosome 3.
Mentions: On the basis of extent of incorporation of BrdU in the presence of HU, Hayashi et al. [15] divided the pre-RCs that they identified into two classes: strong/early (identified by red circles and squares in our Figs. 2 and 6) and weak/late (identified by dark blue circles and squares in our Figs. 2, 6 and 8). As shown in Fig. 3C (based on results in Additional File 7), these two classes corresponded well with our five classes. The origins that we classified as below limit or very weak were mostly weak/late according to Hayashi et al., while the origins that we classified as medium or strong were almost entirely strong/early by their classification [15].

Bottom Line: Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories.We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU.The fact that approximately 97% of fission yeast replication origins - both early and late - are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, SUNY at Stony Brook, Stony Brook, New York 11794-5222, USA. katie.mickle@gmail.com

ABSTRACT

Background: In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results: Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion: The fact that approximately 97% of fission yeast replication origins - both early and late - are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

Show MeSH
Related in: MedlinePlus