Linda Bloom - US grants

DESCRIPTION (provided by applicant) Functional themes in replicases have been conserved from prokaryotes to eukaryotes. DNA polymerases use ring-shaped sliding clamps to achieve the processivity required to replicate a genome. There are remarkable structural similarities in sliding clamps from bacteria, yeast, and man. These clamps are assembled onto DNA by the activity of multisubunit clamp loaders that couple hydrolysis of ATP to the mechanical task of clamp loading. This process requires that protein-protein and protein-DNA interactions change during the clamp loading reaction. Binding and hydrolysis of ATP promote conformational changes in the clamp loader that modulate these interactions. We propose to define the dynamic protein-protein and protein-DNA interactions that are required for the gamma complex clamp loader to assemble the Escherichia coil DNA polymerase III beta clamp onto DNA. Real time fluorescence-based assays will be used to monitor protein-protein and protein-DNA interactions directly in solution and correlate these with ATP binding and hydrolysis. This approach gives our laboratory the unique ability to define changes in the protein-protein and protein-DNA interactions that occur on a millisecond timescale during the clamp loading reaction. We propose to use these experiments to test a kinetic model for clamp loading by defining interactions between the clamp loader and clamp, by defining nucleotide-dependent steps, and by using FRET to measure clamp opening and closing. Much of what has been learned about these replisomes has come from studies of the E. coli system and many analogies are seen in the human system. An E. coil model system offers the advantage that stable proteins can be obtained in high yields for in vitro analysis.

DESCRIPTION (provided by applicant): Duplication of the genome by DNA replication is a prerequisite for normal cell division required for growth and development. Synthesis of DNA is catalyzed by DNA polymerases, however, these enzymes alone cannot make DNA efficiently enough to duplicate the entire genome. DNA polymerase processivity factors, a sliding clamp and clamp loader, enhance the efficiency of DNA replication by tethering a DNA polymerase to the template being copied. The structure and function of these processivity factors are conserved from bacteria to man. The clamp loader is a molecular machine that uses ATP to catalyze the assembly of ring-shaped sliding clamps onto DNA. The major goal of this proposal is to elucidate the mechanism by which the eukaryotic clamp loader, replication factor C (RFC), assembles clamps on DNA by defining functions for individual components. Our overriding hypothesis is that each interaction RFC makes with its binding partners, including individual ATP molecules, the clamp (PCNA), and DNA, induces conformational changes that facilitate the next step in the pathway, and these discrete conformational changes favor an ordered sequence of events to promote efficient clamp loading. Our major approach to testing this hypothesis will be to analyze reactions catalyzed by purified Saccharomyces cerevisiae RFC and an alternative Rad24-RFC clamp loader in vitro using fluorescence-based assays to measure proteinprotein and proteinDNA interactions as well as ATP hydrolysis. In addition, site-directed mutagenesis to conserved sequence motifs in ATP binding sites will be used to evaluate the contributions of individual RFC subunits to clamp loading. Specifically, our aims are 1) to define functions for ATP binding and hydrolysis by individual RFC subunits, 2) to use the alternative clamp loader, Rad24-RFC, as a tool to identify contributions that the large "A-subunit" of RFC makes to PCNA and DNA binding, 3) to identify reciprocal effects of clamp and DNA binding on the activities of RFC and Rad24- RFC. A major strength of our fluorescence approach is that this dynamic clamp loading reaction can be monitored directly in solution and in real time to uncover the temporal order of events, and factors that give rise to this order. Our broad and long-term objectives are to define molecular mechanisms by which the replication machinery duplicates genomes, and to define mechanisms by which these enzymes respond to DNA damage that is encountered during replication. This project will contribute to those objectives by characterizing the biochemical activities of DNA polymerase processivity factors, RFC and PCNA, and of a DNA damage checkpoint complex, Rad24-RFC. A fundamental understanding of the biochemical basis of DNA replication is essential to making clinical correlations between biochemical defects and disease. Basic research in the area of DNA replication has led to the development of important medical diagnostic tools as well as the development of therapeutic agents that inhibit replication of pathogens.

Abstract

Intellectual Merit: DNA replication is the process by which all living organisms make copies of their DNA, and it is the foundation of biological inheritance. The synthesis of the new DNA strands is catalyzed by enzymes called DNA polymerases, which use the mother DNA strand as a template to synthesize a new copy. From bacteria to humans, efficient DNA replication requires proteins known as processivity factors to ensure that the polymerase moves rapidly along DNA without dissociating from it. In particular, sliding clamps are oligomeric ring-shaped proteins that encircle DNA, providing an anchor for the DNA polymerase. To load clamps onto DNA, an open clamp loader-clamp complex must form. It is generally assumed that clamps exist as closed rings in solution and that clamp loaders must therefore actively open their interfaces. Very few studies, however, have addressed this problem directly. The dynamics of clamp opening will be investigated with the goal of understanding the mechanisms by which clamp loaders are able to load sliding clamps onto DNA. The fairly static view of clamp loading that has emerged from structural data is intrinsically inadequate to understand the mechanistic details of how these proteins achieve their function. This limitation will be tackled directly by our experimental design, which is based on the measurement and analysis of the spontaneous fluctuations of a small number of molecules. Initially, the solution oligomerization equilibrium dynamics of the processivity clamps of E. coli (a dimer) and S. cerevisiae (a trimer) will be characterized. These proteins are among the most studied sliding clamps, and yet their association affinities and rate constants have not been fully characterized. Then, the conformational dynamics of sliding clamps in solution, bound to the clamp loaders, and bound to DNA will be characterized. Single-molecule fluorescence techniques are particularly well-suited to investigate the structural dynamics of biopolymers, and will be used in this project to characterize the conformational fluctuations in processivity clamps. The successful completion of these studies will provide vital mechanistic insights into how processivity factors work.

Broader Impacts: Graduate students can enrich their educational experience by learning about and participating in all aspects of the synergistic and joint efforts of the Levitus (ASU) and Bloom (UF) labs. ASU students will spend a fraction of each summer at UF to immerse themselves into the molecular biology aspects of the project, while a student from UF will spend time at ASU to learn about single-molecule and other spectroscopic techniques. Students from underrepresented groups will be recruited through a series of existing programs at ASU and UF. A series of activities aimed at increasing the retention and chances of success of minority students and early-career faculty, including mentoring female junior faculty and minority graduate students, will be continued, as well as participation in student research conferences for underrepresented undergraduate students within the STEM disciplines.

PROJECT SUMMARY Antibiotic resistance is a serious problem in the United States and around the world, particularly in hospitals. In the U.S. alone, millions of people acquire antibiotic-resistant infections each year, and thousands die from these infections. Although antibiotic resistance can be controlled to some degree by surveillance programs that track resistance and by establishing strict guidelines for appropriate antibiotic use, the natural selection process cannot be stopped and resistance will ultimately emerge. Therefore, it is imperative that we maintain a pipeline of new antibiotics and develop antibiotics that act on novel drug targets. Our project addresses these critical needs by defining the mechanism of a naturally occurring bacteriostatic inhibitor of Escherichia coli. N4gp8, a polypeptide produced by bacteriophage N4, binds the DNA polymerase clamp loader and rapidly shuts down DNA replication by inhibiting clamp loading. Because clamp loaders are absolutely required for DNA replication in all bacteria, and consequently for cell growth and proliferation, the clamp loader represents an attractive, and to date unexploited, drug target. Our overarching hypothesis is that N4gp8 presents a model for the design of a new class of antibiotics that act on a novel target. This project will define the mechanism of action of N4gp8 to inform future efforts to develop clamp loader inhibitors. The clamp loader catalyzes the assembly of ring-shaped sliding clamps on DNA, and in doing so, must bind three ligands, ATP, the clamp, and DNA and undergo ligand-induced conformational changes that drive the mechanical reaction. Therefore, there are multiple mechanisms by which N4gp8 could function. This proposal will define the mechanism of inhibition of clamp loading by N4gp8 by defining which clamp loader activity is inhibited in Aim I and by defining molecular interactions between the N4gp8 and the clamp loader that are required for inhibition activity in Aim II. Assays that we developed to investigate clamp loader mechanism will be used along with standard binding assays to identify the step in the clamp loading reaction that is inhibited by N4gp8 and to measure N4gp8-clamp loader binding constants. A combination of techniques will be used to characterize the solution structure of N4gp8 and its active oligomeric state, as well as to define the binding site for N4gp8 on the clamp loader and key protein-protein interactions required for inhibition. These mechanistic studies will elucidate structure-function relationships that can be used in the rational structure-based design of new clamp loader inhibitors, and in developing screens for inhibitors that function by a N4gp8-type mechanism. The clinical relevance of the E. coli model system used here is high because E. coli is a member of the Enterobacteriaceae family of Gram-negative bacteria, and carbapenem-resistant Enterobacteriaceae are classified by the CDC as an urgent antibiotic resistance threat.

Mechanisms of SSB in Coordinating DNA Replication and Repair

When enzymes duplicate chromosomal DNA or repair DNA damage, the DNA duplex is unwound and regions of single-stranded (ss) DNA are exposed. Single-stranded DNA binding proteins (SSBs) play a key role in protecting DNA from damage by binding ss DNA that forms during normal cellular processes. This project addresses the questions of how SSBs bind ss DNA tightly to protect it, but at the same time allow key enzymes to access the DNA to perform their necessary activities. A combination of biochemical and biophysical techniques will be used to investigate the interactions between SSB and three different enzymes that help replicate DNA or repair DNA damage. This project will also be used as a platform for teaching scientific concepts and techniques to students of all ages in order to foster their career development. Students ranging from high school to graduate school will be actively engaged in performing the experiments. More experienced students will be encouraged to mentor newer students, thus fostering their teaching and mentoring skills, as well as solidifying their own core knowledge. The students who work on the project will also develop YouTube video vignettes that use entertaining ways to explain the science behind the project to younger students to excite young students about science and to encourage more young people to explore STEM-related career opportunities.


Single-stranded DNA binding proteins (SSBs) bind single-stranded (ss) DNA intermediates of DNA metabolism to protect ssDNA from damage and to prevent the formation of potentially mutagenic DNA secondary structures. Another critical function of SSBs is to mediate genome maintenance pathways by physically and functionally interacting with many different enzymes. These basic functions of SSB are conserved in all domains of life, but they would seem to interfere with one another. This project uses an Escherichia coli model system to uncover general mechanisms by which SSB and enzymes cooperate to enhance the efficiency of enzyme-catalyzed reactions on DNA. Despite their critical importance, mechanisms by which SSB-enzyme interactions stimulate enzyme activity and allow enzymes to gain access to DNA are not fully defined. Moreover, few studies have measured both enzyme stimulation and DNA-SSB remodeling under the same kinetic conditions to determine how the two processes are linked. This research addresses these questions by: 1) defining mechanisms by which E. coli SSB stimulates enzyme activity, and 2) defining dynamic interactions between enzymes and DNA-SSB that give enzymes access to DNA substrates. Interactions of three model enzymes, a clamp loader, a DNA polymerase, and a DNA helicase, with SSB will be investigated. These enzymes were chosen because each acts on the same DNA substrate, and thus will encounter the same dynamic DNA-SSB structure. This will increase the impact of the findings by permitting identification of mechanisms that are common to all three enzymes, and those that may be specific for a given enzyme.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.