The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Our laboratory focuses on two regulatory aspects of DNA replication -- the regulation of initiation of chromosomal replication and the possible regulation of transcription by DNA replication.

Defining a Chromosomal Origin of Replication. DNA replication is perhaps the most precisely regulated protein-nucleic acid interaction in biology; each base pair of each chromosome is replicated once per cell cycle and only once per cell cycle. Much of this cell cycle regulation is likely to occur at origins, the chromosomal sites at which replication initiates. Little is known about the molecular mechanisms that control initiation of replication and couple replication to the cell cycle because chromosomal origins are ill-defined.

We are currently defining the sequences that comprise a chromosomal origin of replication in the yeast Saccharomyces cerevisiae. Recently, a two-dimensional electrophoretic technique has been developed that allows us to determine directly whether any particular region of a yeast chromosome contains an origin of replication. We have used this technique to identify an origin at the HMR locus, a region involved in determining yeast mating-type. By creating mutations in this region of the chromosome we can identify DNA sequences that are required for initiation of replication. So far, we have identified two small sequence elements that are required for initiation of chromosomal replication. One element appears to be uniquely dedicated to replication, whereas the other element is a transcription factor binding site. This observation suggests that chromosomal replication and transcription share common regulatory mechanisms.

Role of DNA Replication in Regulating Transcription. Our analysis of the HMR chromosomal origin has revealed a second connection between DNA replication and transcription. Silencers are regulatory control elements that can simultaneously repress transcription of multiple genes throughout a region of a chromosome. This type of regional regulation of transcription occurs in all eukaryotes, and is the basis for phenomena such as X-chromosome inactivation in mammals and position effect variegation. To date, however, it has only been possible to identify the DNA elements that direct this type of transcriptional regulation, silencers, in yeast. Our analysis of replication demonstrated that the chromosomal origin of replication that we study is part of a yeast silencer. Our current hypothesis is that the origin is a critical feature of the silencer and that initiation of replication from the silencer is required to repress transcription in the surrounding chromosomal region.

Genetics of Replication and Silencing. We are beginning to identify the proteins that regulate initiation of DNA replication and silencing. If initiation of DNA replication from the silencer is required to repress transcription of the genes at HMR, the proteins that regulate replication must also regulate silencing. We are exploiting this relationship between silencing and replication to identify genetically the genes that regulate replication. We have developed a simple and very sensitive colony-color assay that allows us to isolate genes that regulate silencing. Our preliminary results suggest that some of these genes also control initiation of replication. Our long term goals are to identify the genes that impose cell cycle regulation on the initiation of DNA replication and to determine the role that DNA replication plays in regulating transcription of chromosomal regions.