I have been in love with DNA for as long as I can remember. I chose to study replication because it is central to the biological role of DNA as the molecule of inheritance. One of the most striking features of replication in eukaryotic cells is the precision with which each and every chromosomal DNA molecule is replicated exactly once per cell cycle. As a graduate student I demonstrated that this control is also exerted on the naturally occurring, multiple-copy 2-micron plasmid in the yeast Saccharomyces cerevisiae. My current work has shifted to yeast chromosomes and their origins of replication--the sites in DNA where replication begins.
Research is conducted in collaboration with Research Assistant Professor M. K. Raghuraman (Raghu). We are studying the regulation of replication that ensures that each chromosome is duplicated in a timely and precise way. Although the chromosomes of S. cerevisiae (average size, 800 kb) are orders of magnitude smaller than those of plants and animals, they are organized for replication in much the same way: replication occurs from multiple, closely-spaced origins and different parts of a chromosome are replicated at different times during the S phase of the cell cycle. Early on, we developed 2-dimensional gel electrophoresis techniques that allow us to map specific replication origins and to determine the efficiency with which they are activated. More recently, we have developed methods and algorithms to study replication on a genome wide scale using microarrays. The combination of these techniques, along with the tractability of the yeast genome, has allowed us to pose important questions about DNA replication in eukaryotes.
Due to the semi-discontinuous nature of replication, each active replication fork will necessarily generate a stretch of single stranded template on the lagging strand. In the presence of a drug (hydroxyurea) that inhibits nucleotide synthesis these single stranded regions persist and increase in length. In cells containing a rad53 checkpoint mutation, treatment with hydroxyurea causes replication forks to remain in the immediate vicinity of origins of replication. Therefore, we reasoned, if there were some way to map the locations of these single stranded regions, it would be possible to infer the locations of origins of replication. To map the locations of single stranded regions in the genome, we isolated DNA from these cells and used it as the template for in vitro synthesis using labeled nucleotides. Since we did not denature the genomic DNA, the only regions that can serve as templates for incorporation of the labeled nucleotides are exactly the regions that were single stranded in vivo. We then mapped the locations of these single stranded regions on a genome-wide scale by hybridizing the labeled DNA to microarrays. In a single experiment we were able to map the locations of all S. cerevisiae origins. This powerful technique has been applied to another yeast, S pombe, and we hope to expand our studies on origin identification in other species, including humans. We are also working to understand the role of Rad53 protein at the replication fork.
In wild type cells, each origin appears to have a preferred time within S phase when it becomes actively engaged in replication. Using the tried-and-true Meselson/Stahl experiment we can monitor when in S phase different regions of the genome replicate—that is, make the transition from having two old strands (Heavy-Heavy or HH DNA) to having one old and one new strand (Heavy-Light or HL DNA). DNA is purified from cells at different times in S phase, and the HH and HL DNAs are separated, differentially labeled, and co-hybridized to microarays to determine which parts of the genome have replicated. In the absence of one of the key cell cycle regulators, Clb5 (an S phase cyclin) much of the genome is slow to replicate and the defect appears to be in the activation of origins that normally initiate replication late in S phase. We are currently exploring the nature of this origin activation defect by physically tethering different Clb proteins directly to the inactive origins to identify which cyclins can substitute for the missing Clb5 protein. We are also interested in understanding why late replicating origins are uniquely dependent on Clb5 for activation while early origins are not. This distinction may shed new light on how and why cells activate different origins at different times throughout S phase.
Genomic sequences have recently become available for yeast species related to S. cerevisiae. These species show syntenic blocks of genes that have been dispersed throughout the genome over evolutionary time by inversion and translocation events. We hope to study the replication of chromosomes in these other species using our 2-D gel and microarray approaches to identify their origins of replication and to characterize their temporal pattern of activation. This information will help us answer questions about the evolution of origin sequences, the extent to which origin locations are conserved, and the importance of the temporal program of replication. In particular, we are interested in what ways genome architecture may be shaped by the requirements of origin locations and replication dynamics.
Feng, W, Collingwood, D, Boeck, M. E., Fox, L. A., Alvino, G.M., Fangman, W. L., Raghuraman, M. K., Brewer, B. J. 2006. Nature Cell Biology 8:148-55. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.
Vogelauer, M., Rubbi, L., Lucas, I., Brewer, B. J, and Grunstein, M. 2002. Histone acetylation regulates the time of replication origin firing. Molecular Cell 10:1-11
Raghuraman, M. K., Winzeler, E. A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart, D. J., Davis, R. W., Brewer, B. J., and Fangman, W. L. 2001. Replication dynamics of the yeast genome. Science 294:115-121.
van Brabant, A. J., Buchanan, C. D., Charbonneau, E., Fangman, W. L., and Brewer, B. J. 2001. An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Molecular Cell 7:705-713.
Donaldson, A. E., Raghuraman, M. K., Friedman, K. L., Brewer, B. J. and Fangman, W. L. 1998. Clb5 is required for the timely activation of late replication origins in yeast chromosomes. Molecular Cell 2:173-182.
Raghuraman, M. K., Brewer, B. J., and Fangman, W. L. 1997. Cell cycle-dependent establishment of a late replication program. Science 276:806-809.
Friedman, K. L., and Brewer, B. J. 1995. Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods in Enzymology 262:613-627.
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