In all living organisms, the biosynthesis of DNA (during cell division or chromosome repair) requires four building blocks, collectively called deoxyribonucleoside triphosphates (dNTPs). Chemically, the dNTPs (also called deoxyribonucleotides) are phosphorylated derivates of the four corresponding deoxyribonucleosides (deoxyguanosine, deoxyadenosine, deoxycytidine and thymidine). The biological synthesis of dNTPs is absolutely dependent on an enzyme (ribonucleotide reductase, RNR), which reduces ribonucleotides to the corresponding deoxyribonucleotides. This reaction is essential for the de novo synthesis of DNA, and so RNR is an essential enzyme in virtually all organisms. The activity of RNR and the expression of the genes encoding the two subunits of the enzyme are tightly regulated. These regulatory mechanisms ensure that the cell is provided with a balanced supply of the four dNTPs, and that the pool of dNTPs is matched to the requirements of DNA synthesis and repair. Bacteria typically produce more than one type of RNR. For example, in the model bacterial species Escherichia coli, there are three RNRs: NrdAB requires molecular oxygen and is essential for growth under aerobic conditions; NrdDG is oxygen-independent and essential for anaerobic growth; NrdEF is expressed in response to various stresses and may be required to supply dNTPs for DNA repair processes. The genes encoding these three enzymes are all subject to negative regulation by a transcriptional repressor designated NrdR. It is believed (though not proven) that NrdR represses expression of the RNR-encoding genes in response to high levels of dNTPs. Thus, NrdR is likely to be a key component of a homeostatic feedback loop, in which production of the RNRs is down-regulated when cellular dNTP concentrations are high. In this project, the mechanism of the E. coli NrdR protein will be investigated. The affinity of NrdR for different nucleotides and deoxynucleotides will be measured, and their effects on the DNA-binding activity of NrdR evaluated. Structural properties of NrdR, and the complex that it forms with DNA from the promoter regions of the RNR-encoding genes will be investigated by electron microscopy and by X-ray crystallography. The contribution that NrdR and other factors make to the regulation of expression of NrdEF by oxidative stress will also be explored. Collectively, these studies will lead to a deeper understanding of the mechanism of NrdR, and the role that it plays in controlling the expression of the genes encoding RNRs.
Broader impacts
The project will further understanding of a fundamental biological process, that is the biosynthesis of the monomeric precursors of DNA, an essential prerequisite to chromosome replication and cell proliferation. The research will involve the training and teaching of post-doctoral, graduate and undergraduate researchers. Undergraduates will have opportunities to participate directly in laboratory research, by taking credit-bearing research courses, and through participation in Institute-funded research opportunity programs. There will also be opportunities for high school students participating in summer semester research opportunity programs. The principal investigator participates in programs aimed at establishing mentoring relationships with high school teachers and students, and programs that mentor and provide research experiences for microbiologists from underrepresented groups, and will develop outreach activities in a local elementary school. Results of the research will be published in international peer-reviewed journals, and will be disseminated at national and international conferences. As appropriate, results will also be published in non-technical literature and online, and will be disseminated to the broader community with the help of the UT Dallas Office of Communications. Graduate and undergraduate students will attend meetings of professional societies, and will present oral and poster communications of the results of their research. The Texas Branch of the American Society for Microbiology is extremely active, meets twice a year, and provides a supportive environment in which graduate and undergraduate researchers can present their results and network with peers and other academic scientists. Graduate students will be encouraged to participate in the teaching of undergraduates and high school students, by acting as Teaching Assistants in undergraduate courses and as mentors in the lab.
