Education:

BS (1979) UC Davis, Biochemistry and Biophysics
PhD (1992) UCLA, Biological Chemistry
Postdoctoral Fellow (1992) Stanford University Medical School, Biochemistry

Research Interest

Chromosomal replication is regulated at the initiation stage, a key control point in the determination of eukaryotic cellular quiescence or proliferation and the prokaryotic cell cycle. In the most general terms, initiation involves initiator protein(s) acting at an origin of replication on the chromosome. The host-encoded initiator protein in E. coli is DnaA protein. Understanding the regulation of DnaA protein should provide valuable insight into the initiation of replication in eukaryotes, especially with the recent finding that bacterial DnaA and eukaryotic Cdc6 protein have strikingly similar structures. Regulation of DnaA protein activity in E. coli appears to be a key step for controlling the start of DNA replication. Since the cellular abundance of DnaA is largely constant throughout the cell cycle, other mechanisms must regulate the protein's activity. Prominent among these is the influence on the replicative-action of DnaA by the tight binding of ATP and ADP. Both the ADP and ATP forms of DnaA bind the origin of replication, oriC, but only the ATP form is active for subsequent replication steps. The ADP form fails to promote strand opening and is inert for replication. Recently it has determined that a specific cis-acting element in the origin serve as the discriminator between ADP- and ATP-DnaA. Acidic membranes have been observed to reactivate ADP-DnaA in vitro via a rapid exchange of ADP and ATP, generating fully replicative-active ATP-DnaA needed for initiation of chromosomal replication for the next cell-cycle. Genetic and physiological studies further suggest a close link between membrane lipid composition and initiator activity in chromosomal replication. Confocal fluorescent microscopy imaging of GFP-tagged DnaA in live cells suggests that DnaA interacts with membrane-associated cytoskeletal structures, and therefore that spatial as well as temporal regulation is important. Also, recent findings further reveal that the conversion of ATP-DnaA to ADP-DnaA is critical for the production of deoxynucleotides by triggering the transcription of ribonucleotide reductase at the onset of chromosomal DNA synthesis. Overall, the long-term goal of these studies is to elucidate the physiological significance of modulating the activity of an initiator protein in the control of chromosomal replication.

A second project in the laboratory focuses on understanding the physiological role that cellular inorganic polyphosphates play in how cells respond to environmental stresses. Cellular polyphosphates are dynamic, long linear polymers of orthophosphates linked by high-energy phosphoanhydride bonds. They are ubiquitous in nature, having been found in every tissue of every organism examined, including human breast cancer cells. Studies with numerous microbes have revealed that cells that are void of polyphosphates are highly susceptible to killing when exposed to cytotoxic agents, such as cisplatin and mitomycin C, or treated with UV or ionizing radiation. Work with E. coli indicates that the poor survival is because the cells lacking polyphosphates are compromised in how they carry out error-prone DNA repair. Further work to define the molecular mechanism linking polyphosphates and the action of translesion DNA polymerases is ongoing. Similarly, breast cancer cells that have been depleted of their polyphosphates through expression of the S. cerevisiae polyphosphate-degradative enzyme, exopolyphosphatase (ScPPX), are known to have much poorer survival than non-modified cells when cultured in serum-free medium, suggesting that polyphosphates play an important role in the survival of breast cancer cells when exposed to challenging conditions. A goal of our research is to determine whether breast cancer cells that have been depleted of their polyphosphates are substantially sensitized to killing by established chemotherapeutic and radiation treatments. Ultimately, targeting the enzyme responsible for polyphosphate biosynthesis in breast cancers could facilitate the desired killing of breast cancer cells at lower dosages of radiation and chemotherapeutics, thus minimizing their deleterious side effects while maintaining maximal efficacy.