Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork - PubMed (original) (raw)
Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork
Arturo Calzada et al. Genes Dev. 2005.
Abstract
Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.
Figures
Figure 1.
Using the Fob1–RFB system to pause specific DNA replication forks on chromosome 3. (A, panel i) Map of two consecutive repeats of the rDNA on chromosome 12 of budding yeast. The positions of the replication fork barrier (RFB), origin of DNA replication (ARS), 35S rRNA (35S), and 5S rRNA (5S) genes are indicated. (Panel ii) Insertion of RFB sequences on chromosome 3 to block the forks from ARS305 and ARS306. The distances indicated are measured from the left end of chromosome 3; “B” and “S” mark the positions of BclI and SalI restriction enzyme sites used for the two-dimensional DNA gels. See Materials and Methods and Supplementary Figure 1 for more information. (Panel iii) Using two-dimensional DNA gels to study DNA replication intermediates; see text for details. The two spots below the Y-arc in the left panel correspond to other sites in the genome that are recognized weakly by the chosen probe. (B) DNA replication forks do not pause at the RFBs on chromosome 3 in the absence of Fob1. (C, panel i) In cells expressing Fob1, the forks from ARS305 and ARS306 pause for an extended period at the two RFBs on chromosome 3. (Panel ii) The histograms show a quantification of paused forks at the indicated times (calculated as described in Materials and Methods).
Figure 2.
Pausing of forks at the RFBs does not cause the replisome to disassemble. (A) ChIP was used to study the localization of proteins at the five sites indicated. Further details can be found in Supplementary Figure 4. (B) Cells were synchronized in the G1 phase of the cell cycle before expressing GAL-FOB1-9MYC for 45 min, maintaining the G1 arrest throughout. The Fob1-9Myc protein was detected specifically at the two RFBs introduced on chromosome 3. (C [panel _i_], D) The localization of Pol2-9Myc or Psf2/Cdc102-9Myc was examined at the same five sites, as cells entered synchronously into the S phase of the cell cycle in the presence (+RFBs) or absence (–RFBs) of the Fob1–RFBs on chromosome 3; see text for details. (C, panel ii) The region between ARS305 and the corresponding RFB was also examined with higher resolution.
Figure 3.
Other components of the paused replisome. (A, panel i) Cdc45 accumulates during S phase at the sites of the paused forks (+RFBs) but is more transiently associated with the same sites in the absence of pausing (–RFBs). (Panel ii) Mcm4, Mrc1, Tof1, and Pri1 all remain associated with the forks that pause at the Fob1–RFBs on chromosome 3. (B) The localization of Pri1-9Myc and Cdc102-5Flag was determined in the same cells at the indicated times. The change in the specific enrichment of the RFB sequences is shown from 30 min (100%) to 40 min. (C) Rrm3 is specifically recruited to paused replisomes (panel i), and is not a stable component of active forks established at origins of replication (panel ii).
Figure 4.
Mrc1 is not essential for DNA replication forks from ARS305 and ARS306 to pause at the RFBs on chromosome 3, but Tof1 and Csm3 are both important. Fork progression was examined as above in cells lacking Tof1 (A), Mrc1 (B), or Csm3 (C). (D) Larger versions of the data from the 60-min time point. (E) Fob1 still associates with the RFBs on chromosome 3 in the absence of Tof1 or Csm3. Cells were synchronized in G1 phase, and expression of Fob1-9Myc was then induced for 45 min.
Figure 5.
Pausing of the replisome at the RFBs on chromosome 3 is independent of Mrc1 but requires Tof1. The association of Cdc45 with the region between ARS305 and ARS306 was examined by ChIP at 30 min or 45 min after release of cells from G1 arrest.
Figure 6.
(A) Mrc1 is not essential for DNA replication forks to pause at the endogenous RFB within the rDNA, but Tof1 and Csm3 are both important for pausing. (B) Larger versions of the data from the 45-min time point. The arrows mark the site where forks normally pause at the RFB (1), as well as a region beyond this point where weak pausing can be seen in the absence of Tof1 or Csm3 (2).
Figure 7.
Stability of forks that pause at the RFBs on chromosome 3 does not require checkpoint kinases. Progression of forks was examined as before in _sml1_Δ (A), _mec1_Δ _sml1_Δ (B, panel i) or _rad53_Δ _sml1_Δ (B, panel ii) strains.
Figure 8.
Stability of paused forks at the RFB in the rDNA is independent of checkpoint kinases. (A) Progression of forks was examined as before in _sml1_Δ, _mec1_Δ _sml1_Δ, or _rad53_Δ _sml1_Δ strains. (B) Larger versions of individual time points are shown for the three strains.
Figure 9.
Recovery of DNA synthesis from forks paused at the Fob1–RFB does not require recombination. (A, panel i) Viability of the indicated strains was measured as cells were released from G1 phase in the presence of Fob1, as in previous experiments. DNA content was measured by flow cytometry. (Panel ii) Growth of cells lacking Rad52 is not affected by extended pausing of DNA replication forks at the RFBs on chromosome 3. Serial dilutions of cells were plated on YPD medium (inactive RFBs) and YPGal medium (active RFBs) and photographed after 48 h growth at 25°C. Note that yeast cells grow more slowly on YPGal medium. (B) Progression of DNA replication forks through the region between ARS305 and ARS306 was examined as before. (C) Replication of the rDNA repeats was examined in the same experiment.
Figure 10.
A model for the pausing and recovery of DNA replication forks at a protein–DNA barrier; see text for details.
References
- Alcasabas A.A., Osborn, A.J., Bachant, J., Hu, F., Werler, P.J., Bousset, K., Furuya, K., Diffley, J.F., Carr, A.M., and Elledge, S.J. 2001. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 3: 958–965. - PubMed
- Allen J.B., Zhou, Z., Siede, W., Friedberg, E.C., and Elledge, S.J. 1994. The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes & Dev. 8: 2401–2415. - PubMed
- Aparicio O.M., Weinstein, D.M., and Bell, S.P. 1997. Components and dynamics of DNA replication complexes in S. cerevisiae: Redistribution of MCM complexes and Cdc45p during S phase. Cell 91: 59–69. - PubMed
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