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Professional Interests - Publications - Contact Information - Lab Personnel Professional Interests of Anthony Schwacha Chromosome replication and the subsequent segregation of sister chromatids into daughter cells are fundamental molecular processes that define life. Chromosome replication involves not only duplication of the genetic information present on each of the component DNA strands, but also the precise replication of its functional state. This incredibly complex process must be performed precisely every cell cycle: mistakes in replicating the DNA result in a loss or alteration of the genetic code, while changes in chromosome structure can alter the expression of the component genes and the ability to segregate properly during mitosis. The net result is either case is often cellular death or a predisposition to further genomic alterations that can lead to carcinogenic state. Much remains to be understood not only in the details of DNA replication, but also how this process is regulated and integrated into the structure and function of the nucleus.
I study two areas of eukaryotic chromosome replication utilizing the yeast Saccharomyces cerevisiae. The first is to obtain a mechanistic understanding of how DNA replication is initiated and regulated. As an entry point into addressing this problem, my attention has focused on the minichromosome maintenance (MCM) complex, the only eukaryotic replication factor that is required for both the initiation and elongation stages of DNA replication. All eukaryotes contain six essential MCM genes. These genes are partially homologous with one another and each contain ATP binding and hydrolysis motifs. Together, the products of these six genes form a heterohexameric ATPase. By studying a large variety of heterohexameric complexes containing specific combinations of either wildtype or mutant subunits, I've demonstrated unusual allosteric regulation within the complex: 3 are responsible for ATP hydrolysis, while the other 3 appear to have a regulatory function. Kinetic analysis of the ATP hydrolysis of both wildtype and mutant MCM complexes extends these observations, and suggests an unexpected mechanistic parallel between the MCM complex and an unusual but well studied protein known as the F1-ATPase. The F1-ATPase is part of a large protein complex that functions in the key step of the process that generates cellular ATP from ingested food. However, unlike the MCM complex, the mechanistic role of ATP hydrolysis by this complex is well known: elegant single-molecule experiments demonstrate that the F1-ATPase functions as a rotary motor. Binding and hydrolysis of each ATP molecule causes a 120o rotation of a "driveshaft" protein that functionally couples the activity of the F1 ATPase to additional parts of the complex. By analogy, this suggests that perhaps the MCMs - as well as other proteins such as helicases - function as a rotary motor, perhaps utilizing either double stranded or single stranded DNA as a "driveshaft" to cause unwinding or pumping of DNA. I am currently pursuing two general questions concerning the MCM complex; 1) is it actually a rotary motor, and 2) what is its actual in vivo function during DNA replication? A variety of biochemical, molecular, and genetic approaches are being utilized to address these questions.
My second research area involves the spatial regulation of DNA replication. Cytological evidence from both prokaryotes and eukaryotes indicates that, unexpectedly, DNA replication does not occur randomly throughout the nucleus, but rather at a relatively few foci, implying that multiple replication forks cluster together in vivo to form large DNA replication "factories" that remains stationary while the DNA is threaded through. These observations, as well as similar observations involving other nuclear processes (i.e., transcription, RNA splicing), indicate that the nucleus is not a random bag of DNA, but is functionally organized. Despite the implied important relationship between nuclear structure and function, little work has been attempted to understand its molecular basis. Using the yeast S. cerevisiae, a cytological screen will be used to identify mutations that interfere with this process. These mutations will thus define essential components in this process, provide clues concerning its biological function, and allow a start to the biochemical analysis of this phenomenon.
Publication
Archive Recent Publications of Anthony Schwacha
Bochman, M.L., and A. Schwacha (2008) The Mcm2-7 complex has in vitro helicase activity. Mol. Cell 31:287-293 How to Contact Anthony Schwacha
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Fig. 2. Many kinetic and structural considerations suggest possible mechanistic parallels between the F1-ATPase and the MCM complex. In the case of the well-studied F1-ATPase, ATP binding and hydrolysis causes rotation of a protein located in the central cavity of the complex. By analogy to the F1-ATPase, perhaps the MCM complex is able cause rotation of a centrally located substrate in the form of single- or double-stranded DNA, to function as either a helicase to unwind DNA or as a type of DNA "pump" [
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