The Schumacher Lab's Research Focus
The overarching goals of the Schumacher are to determine the molecular mechanisms that control fundamental biological processes involving protein-nucleic acid interactions. The main projects underway in the lab focus on DNA segregation, transcription and RNA editing.
Since her thesis work deciphering the allosteric DNA binding mechanism of LacI proteins, Dr. Schumacher has been fascinated by the structural and biochemical mechanisms that underpin transcription regulation. The lab now primarily focuses on global transcription regulatory networks and systems that control entire developmental processes. These studies have led to the delineation, in atomic detail, of the machinery controlling carbon and nitrogen utilization in model bacteria and more recently the determination of the structure of a fully assembled promoter complex.
Recent studies on kRNA editing are focused on deducing the functions and structures of unique RNA editing accessory factors, which are required for this unusual event, but which are not involved in the catalytic steps of the process. Our studies have explained the basis for RNA matchmaking between gRNA and pre-mRNA. More recent studies have uncovered accessory proteins with potential processivity and gRNA/mRNA specific capabilities. These findings may lead to an explanation for the high degree of accuracy and processivity of this process.
A primary area of interest in the Schumacher lab is DNA segregation or partition. DNA segregation is one of the most fundamental processes in biology. Indeed, the faithful inheritance of genetic information is necessary for the survival of all life as it ensures that every daughter cell receives a copy of genomic or plasmid DNA. The molecular components of this process have been identified in prokaryotes and eukaryotes, however, the detailed mechanisms involved are still unresolved. Our overarching goal is to determine the structural mechanisms behind the basic segregation machinery employed by the three domains of life. We have largely focused on bacterial partition (par) systems. These systems require only three components, making them ideal model systems for atomic level study. These three elements, which are encoded on the plasmid, include a DNA centromere, centromere-binding protein (CBP) and an NTPase (nucleoside triphosphatase). The general steps in prokaryotic plasmid partition involve the binding of multiple CBPs to the centromere to form "partition complexes", which then recruit and stabilizes the formation of NTPase polymers. The NTPase oligomers then somehow drive separation of replicated DNA to cell poles. There are three types of prokaryotic plasmid par systems, called type I, II and III. When we initiated our studies nothing was known structurally about these systems. Over the past ~7 years (since obtaining tenure) the work in my lab has provided a near-complete atomic level delineation of these systems. We have obtained structures for all components of each system and most of the higher order complexes. A particularly groundbreaking structure, which we obtained, was that of the first full-length partition complex, which revealed that it forms a large superhelical structure that caps and stabilizes the growing NTPase actin-like filaments. This led to an atomic level insertional polymerization or "pushing" model for type II DNA segregation, which was supported by cryo-EM, biochemical and in vivo studies. Our combined studies on the three par systems revealed that despite the fact that all use the same three component organization, they employ NTPases with different cytoskeletal folds: either an actin-like, tubulin-like or Walker box fold. As would be expected given their distinct cytoskeletal NTPases, the three par systems use different mechanisms; type I systems "pull", type II systems "push" and type III modules "tram/transport" plasmids (Fig. 1). Since making these discoveries, we have moved on to examine DNA segregation in archaea and eukaryotes. Surprisingly, nothing was known about archaeal DNA segregation. Our recent work on an archaeal segregation system suggests that it may be a bacterial/eukaryotic hybrid; it harbors an NTPase with a bacterial Walker box fold and a centromere binding protein with structural similarity to the eukaryotic histone H3 homologue, CenpA. This is notable because CenpA is specifically deposited at centromere sites, where it replaces histone H3 to "mark the spot" as the assembly site for accretion of the eukaryotic segregation machinery. In addition to completing these studies we are working to obtain structures of the core eukaryotic segregation complex, which includes CenpA and CCAN proteins that comprise the inner kinetochore.
Finally, while our studies on DNA segregation, transcription and RNA editing have provided major advances in these fields, we have also made significant contributions to several other areas of study. These include multidrug resistance/tolerance, ion channel gating, DNA organization, cytoskeletal processes, DNA replication and signaling (e.g. myokine and calcium regulated signaling).
Fig. 1. Schematic models for types I, II and III plasmid partition. Type I partition utilizes the host cell nucleoid as a "track" for NTPase-ATP binding and polymerization (square). When the NTPase-ATP polymer encounters a ParB-centromere partition complex (shown as a circle), that is, the ParB attached plasmid, the NTPase activity is activated resulting in dissociation of capping ParA-ADP subunits (triangles) and polymer retraction. The ParB-plasmid is either pulled along in the retreating ParA polymer or is attracted and diffuses toward the moving polymer resulting in equidistribution of ParB-plasmids at opposite ends of the nucleoid. Type II partition uses a pushing or insertional polymerization mode of segregation. In this model, the dynamically unstable ParM filaments are stabilized and propagate only when each end is captured by a ParR-centromere partition complex. The polymer continues to grow upon addition of ParM-ATP or ParM-GTP subunits to the ParR-ParM +interface. The outcome is redistribution of replicated plasmids to opposite poles. Type III partition employs a tram mechanism of partition. TubR binds the centromere serving as a high local concentration of binding sites for the C-terminal flexible domains emanating from treadmilling TubZ filaments. Once captured, the TubR-plasmid is transported to the cell pole by the treadmilling TubZ filaments. Upon reaching the membrane the TubZ filament bends, likely dumping its TubR-plasmid cargo, and reverses direction. Now traveling in the opposite direction, the TubZ filament binds another TubR-plasmid cargo and carries it to the opposite pole.