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Forsburg Lab Research: MCMs and Genome Stability

Much of our interest of late has been in determining how the panel of molecules in which we are interested, primarily but not only MCMs, contribute to overall genome stability (not just DNA synthesis). A substantial part of this investigation has been based on sophisticated imaging methods on both live and fixed cells.
MCMs are required for fork stability. Years ago, Debbie Liang (a student in the lab) made two important findings. First, she showed that mcm-ts alleles had significant problems even at permissive temperatures, such that reduced dosage of a single MCM protein causes increased rates of chromosome loss and recombination. Second, she showed that the mcm mutants are irreversible: they don't recover if you shift them back down to permissive termperature after an arrest at 36°C, even though they appear to have a 2C DNA content. Thus, the problem isn't in bulk DNA accumulation.

Debbie suggested this was due to DNA damage, because if she deleted the Chk1 damage checkpoint, the cells continued into a lethal mitosis. This can be seen in the phenotypes of the cells as seen on the left below. This was confirmed by Julie Bailis, who showed that mcm mutants accumulate markers of DNA damage, including phosphorylation of histone H2A, and numerous foci of the homologous recombination protein Rhp51.
Damage checkpoint- dependent arrest of mcm2 (from Liang et al, 1999). Wild type cells show a normal cell cycle distribution. mcm2tscells are elongated with a single nucleus due to cell cycle arrest ("cdc" phenotype). However, eliminating the Chk1 kinase allows these cells to proceed into a lethal mitosis, generating a "cut" phenotype.
mcm mutants cause DNA damage (from Bailis et al, 2008). Spread nuclei were stained for markers phosphorylated histone H2A, a marker for double strand breaks. They also accumulate extensive foci of the homologous recombination protein Rhp51 which recognizes a wide range of lesions.
The damage markers that accumulate in mcm mutants are very similar to the markers that accumulate under conditions known to cause replication fork collapse. Normally, the Cds1 checkpoint kinase (Chk2, Rad53 in other systems) is responsible for maintaining the fork for example when cells are starved by nucleotides (treated with the drug hydroxyurea). Cds1 has numerous targets in this process including the DDK kinase (which prevents additional origin firing), the cell cycle machinery, and the endonuclease Mus81. The similarity of the phenotypes of mcm at restrictive temperature, and cds1 + HU, suggested to us that the MCMs are causing fork collapse.

Two additional pieces of evidence supported this finding. First, Julie Bailis found that if she arrested cells in HU, allowing forks to stall, and then inactivated the MCMs (in the continuing presence of HU), that was sufficient to induce damage detected by H2A phosphorylation. That is, without MCMs, the forks collapse. that experiment is shown at right (from Bailis 2008). Julie also showed that one of our mcm4ts alleles was rescued by pre-treatment with HU. Under those conditions, Cds1 is activated, and Doug Luche showed evidence for Mcm4 being phosphorylated in a Cds1 manner in HU, suggesting that MCMs are a proximal target of Cds1 in the maintenance of the replication fork, as diagrammed in the model below. Nimna Ranatunga is now exploring novel Mcm4 alleles to investigate this further.

The architecture of collapse, the strategy of recovery

The work on MCMs led Sarah Sabatinos to ask what REALLY happens during fork collapse? And, even more interesting, what happens during recovery--how are forks restarted? To investigate the dynamics of this process, Sarah has used a unique combination of imaging methods, including single cell analysis of live cells treated with, and then released from HU (this is performed using a microfluidics perfusions system attached to a deconvolution microscope), labeling of new DNA synthesis with thymidine analogues, and analysis of chromatin fibers for a visual architecture of the replication fork. This work has led to some fascinating insights including distinctly different phenotypes in different mutants that by the old "snapshot" methods of analysis appeared indistinguishable. These methods can now be applied broadly to a wide spectrum of replication mutants to distinguish their defects from one another.

Chromatin fiber spreading. Proteinated chromatin fibers are spread from nuclei and analyzed by immunofluorescence for Mcm2, BrdU (labeling new DNA synthesis), and total DNA.

Reviews on genome stability




Our primary research publications on genome stability


  • Singh SK, Sabatinos S, Forsburg S, Bastia D. (2010) Regulation of replication termination by Reb1 protein-mediated action at a distance. Cell 142(6):868-78.
  • Bailis, J.M., Luche, D.D. Hunter, T., and Forsburg, S.L. (2008) MCM proteins interact with checkpoint and recombination proteins to promote S phase genome stability Mol. Cell. Biol. 28:1724-38. PMC2258774
  • Hodson, J.A., Bailis, J.M. and Forsburg, S.L. (2003) Efficient labeling of fission yeast S. pombe by thymidine and BUdR. Nucl. Acids Res. 31:e134. PMC275491
  • Liang, D.T., Hodson, J.A. and Forsburg, S.L. (1999) Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay. J. Cell Sci. 112:559-567


See the complete Forsburg Lab Publication list