Office: 6-106 MCB
Laboratory: 6-212/218 MCB
Lab Telephone: (612) 624-0460
DNA replication is an integral part of the eukaryotic cell cycle, which coordinates the duplication of the entire genome during S phase and chromosome segregation during mitosis. At the onset of S phase, origins of DNA replication are activated to "fire". Once DNA synthesis has been initiated, the cell is committed to undergo a complete round of division. Deregulation of origin activation results, therefore, in uncontrolled cell proliferation, which is at the heart of diseases such as cancer.
To initiate DNA replication, cells must assemble specific complexes, also called replication forks (see cartoon above). Key components of these complexes are the minichromosome maintenance protein 10 (Mcm10) and DNA polymerase-alpha/primase. The latter is the only enzyme in eukaryotic cells capable of synthesizing DNA de novo . This polymerase is thus indispensable for DNA replication. Limiting amounts of it can lead to unstable replication forks and incomplete replication, resulting in chromosome breakage. Therefore, eukaryotic cells have developed a particular mechanism to maintain significant amounts of DNA polymerase-alpha/primase in the nucleus. They stabilize the complex through the interaction with another protein, a nuclear chaperone that is bound to the complex at all times during the cell cycle. This nuclear chaperone is Mcm10. In the absence of Mcm10, the catalytic subunit of DNA polymerase-alpha is degraded in budding yeast (Figure 1) and cells lose the ability to replicate their genome. Mcm10 is conserved from yeast to man, and one focus of our laboratory is to understand the regulation of Mcm10. For further information, please go to http://www.news-medical.net/?id=5743.
Figure 1. Mcm10 is required for stable expression of DNA polymerase-alpha. The catalytic subunit of DNA polymerase-alpha was tagged with GFP and analyzed by fluorescence microscopy. DNA polymerase-alpha accumulates in the nuclei of wild-type cells (top), but is degraded in mutants that are depleted of Mcm10 (bottom).
Another focus in our lab is the investigation of the S phase checkpoint in budding yeast. All eukaryotic cells have evolved various checkpoints to ensure proper cell cycle progression. The S phase checkpoint is activated when replication forks encounter obstacles such as damaged DNA or other impediments that force them to slow down. The S phase checkpoint maintains the stability of replication forks under such circumstances and prevents replication fork collapse, which would also lead to chromosome breakage. S phase checkpoint genes are frequently mutated in cancer cells, and this deficiency might directly contribute to cancer development.
Haworth, J. C., Alver, R., Anderson, M. and Bielinsky, A. K. 2010. Ubc4 and Not4 regulate steady-state levels of DNA polymerase-alpha to promote efficient and accurate DNA replication, Molecular Biology of the Cell 21: 3205-3219. (pubmed)
Nguyen, H. D. and Bielinsky, A. K. 2010. HDM2 ERKs PCNA, Journal of Cell Biology 190: 487-489. (pubmed)
Alver, R. and Bielinsky, A. K. 2010. Termination at sTop2, Molecular Cell 39: 487-489. (pubmed)
Das-Bradoo, S., Nguyen, H. D. and Bielinsky, A. K. 2010. Damage-specific modification of PCNA, Cell Cycle 9: 3674-3679. (pubmed)
Das-Bradoo, S., Nguyen, H. D., Ricke, R. M., Haworth, J. C. and Bielinsky, A. K. 2010. DNA ligase I deficiency triggers PCNA ubiquitination, Nature Cell Biology 12: 74-79. (pubmed)
Das-Bradoo, S. and Bielinsky, A. K., 2009. Mapping replication initiation sites in eukaryotic genomes. Molecular Methods in DNA Replication (ed., John Walker and Jacob Dalgaard), Humana Press Inc.
Raveendranathan, M. and Bielinsky, A. K., 2009. Analyzing origin activation patterns by copy-number change experiments. Molecular Methods in DNA Replication (ed., John Walker and Jacob Dalgaard), Humana Press Inc.
Warren, E., Vaithiyalingam, S., Haworth, J., Greer, B., Smith, J., Bielinsky, A. K., Chazin, W. and Eichman, B., 2008. Structural basis for DNA binding by replication initiator Mcm10, Structure (cover) 16:1892-1901. (pubmed)
Bielinsky, A. K., 2007. Scarce but scary. Nature Genetics 39: 707-708. (pubmed)
Chattopadhyay, S. and Bielinsky, A. K., 2007. Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication. Molecular Biology of the Cell 18: 4085-4095. (pubmed)
Bolon, Y. T. and Bielinsky, A. K. 2006. The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast. Nucleic Acids Research 34:5069-5080. (pubmed)
Raveendranathan, M., Chattopadhyay, S., Bolon, Y.-T., Haworth, J. C., Clarke, D. J. and Bielinsky, A. K. 2006. Genome-wide replication profiles of S phase checkpoint mutants reveal fragile sites in yeast, EMBO J. 25:3627-3639. (pubmed)
Ricke, R. M. and Bielinsky, A. K. 2006. A conserved Hsp10-like domain in Mcm10 is required for the stabilization of DNA polymerase-alpha in budding yeast, Journal of Biological Chemistry 281:18414-18425. (pubmed)
Das-Bradoo, S., Ricke, R. M. and Bielinsky, A. K. 2006. Interaction between PCNA and di-ubiquitinated Mcm10 is essential for cell growth in budding yeast, Molecular & Cell Biology 26:4806-4817. (pubmed)
Bielinsky, A. K. and Raveendranathan, M. 2006. Encircled: large-scale purification of replication origins from mammalian chromosomes, Molecular Cell 21: 735-737. (Pubmed)
Ricke, R. M. and Bielinsky, A. K. 2005. Easy detection of chromatin binding proteins by the histone association assay. Biological Procedures Online, 7:60-69. (Online citation)
Ricke, R. M. and Bielinsky, A. K. 2004. Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. Molecular Cell, 16:173-185. (Medline citation)
Bielinsky, A. K. 2003. Replication origins-why do we need so many? Cell Cycle 2:307-309. (Medline citation)
Gerbi, S. A. and Bielinsky, A. K. 2002. DNA replication and chromatin. Curr. Opin. Genet. Dev. 12:243-248. Medline citation.
Bielinsky, A. K., Blitzblau, H., Beall, E. L., Ezrokhi, M., Smith, H. S., Botchan, M. R., and Gerbi, S. A. 2001. Origin recognition complex binding to a metazoan replication origin. Current Biology 11:1427-1431. Medline citation.
Bielinsky, A. K., and S. A. Gerbi, 2001. Where it all starts: eukaryotic origins of DNA replication. J. Cell Science 114: 643-651. Medline citation.
Bielinsky, A. K., and S. A. Gerbi. 1999. Chromosomal ARS1 has a single leading strand start site. Molecular Cell 3:477-486. Medline citation.
Gerbi, S. A., Bielinsky, A. K., Liang, C., Lunyak, V. V. and Urnov, F. D. 1999. Methods to map origins of replication in eukaryotes in "Eukaryotic DNA Replication: a practical approach" (ed., S. Cotterill), Oxford University Press, pp 1-41.
Bielinsky, A. K., and S. A. Gerbi. 1998. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279:95-98. Medline citation.
Gerbi, S. A., and Bielinsky, A. K. 1997. Replication initiation point mapping. Methods 13:271-280. Medline citation.
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