Tao-shih Hsieh

Professor of Biochemistry

Chromosome structure and function: structure, function, and mechanism of DNA topoisomerases.

Contact Information

Office Number: (919) 684-6501

Fax: (919) 684-8885

e-mail hsieh@biochem.duke.edu

Lab Location

Room 141A

Nanaline Duke Building

Mailing Address

Department of Biochemistry

Nanaline H. Duke

Box 3711, DUMC

Durham, NC 27710

Education

• Ph.D. (University of California, Berkeley, 1977)

Research Interests

The focus of our research program is on the structure, function, and mechanism of DNA topoisomerases, and recQ family DNA helicases. In addition to the biochemical studies, our laboratory uses Drosophila as a system to probe the genetic and biological functions of these enzymes. Our lab has cloned and analyzed the genes encoding all the topoisomerases from Drosophila, including topoisomerases I, II, IIIalpha and IIIbeta. We have generated strains with mutations in topo I, II, IIIalpha, and IIIbeta, and studied the genetics and biological functions of these mutants. Topoisomerases are essential enzymes which can modulate the topological structure of DNA and play important roles in all aspects of chromosome functions including replication, transcription, recombination, repair, and segregation. DNA topoisomerases can allow DNA strands to pass freely through each other, the mechanism by which this is achieved is an important question at the interface of chemistry and biology. Furthermore, DNA topoisomerases are important pharmacological targets for many clinically useful antibiotics and anti-cancer drugs. Studies on the mechanism of topoisomerase can provide new insights into the cancer chemotherapy. These topoisomerase targeting drugs are useful reagents in probing the mechanism and function of these enzymes.

The mechanistic analysis of topoisomerase is centered on two different types of enzymes: topoisomerases II and III. Topoisomerase III is unique among the topoisomerases in that it exhibits low activity in removing either negative or positive DNA supercoils. To address the mechanistic basis of this observation, and the biological functions, of this unusual enzyme, we made novel DNA substrates for studying topo III. We discovered that a topoisomerase from the hyperthermophiles, reverse gyrase, has an important biochemical function as a DNA renaturase. This finding has allowed us to make a number of important new DNA substrates. By annealing two complementary single stranded DNA circles with reverse gyrase, we could make supercoiled DNA with a single stranded bubble (Fig 1). This DNA substrate was used in investigating the biochemical mechanism of both reverse gyrase and topo IIIalpha and IIIbeta. A further modification on this method to make DNA with single stranded bubble has allowed us to synthesize a DNA containing a model double Holliday junction. This novel DNA substrate is composed of two covalently closed circles conjoined by two Holliday junctions in a manner such that the double junction is mobile, capable of undergoing branch migration, and yet it is under topological constraint (Fig 2). This double Holliday junction substrate presents us a useful model molecule for an important intermediate in genetic recombination. To investigate the biochemical roles of various DNA enzymes in resolving this double Holliday junction substrate, we have used resolvase to demonstrate that resolution by this enzyme can result in products with either cross-over or non-crossover of the flanking genetic elements. More importantly, we showed that an enzyme complex of topo IIIalpha, Bloom’s helicase, and single stranded binding protein RPA can resolve the DNA by the mechanism of convergent branch migration (Fig 3). This new finding now opens the door for us to investigate the biochemical mechanism by which this reaction can occur, and to investigate other partners that may affect the outcome of this reaction. We are also interested in an unusual DNA nanomachine, topoisomerase II. Topoisomerase II requires ATP for its strand passage activity between two double stranded DNA segments. We have generated a novel oligonucleotide DNA substrate that contains a topo II cleavage site in the center which is flanked by two fluorophores capable of Fluorescence Resonance Energy Transfer (FRET). Topo II-mediated DNA cleavage and gate opening reduce FRET, providing us with a system to use single molecule experiments to monitor DNA gate opening and closing (Fig 4). Site-specific mutants for the critical residues that can affect this process will be generated and analyzed with both single molecule and ensemble experiments.

A newly developed project in our laboratory is to study the biological and biochemical functions of wuho (wh), which is required for gametogenesis. wuho mutants are male sterile and female semi-sterile. Immunolocalization experiments demonstrate that wh protein is concentrated in the hub cells at the tip of testes (Fig 5). Hub cells are niche cells for germline stem cells . Spermatogenesis in the wh mutant testes is apparently normal up to post-meiotic stages, but specifically arrested at the spermatid elongation stage. The expression of wh is also maximal in the early stages of oogenesis. Mutant ovarioles can have an egg chamber with more than 16 germline cells, suggesting that these cells are not arrested after two cycles of mitotic divisions, and their differentiation into nurse cells are specifically blocked. The cellular defects of wh mutants in oogenesis and spermatogenesis suggest that wh plays an important role in the complex processes of cellular differentiation. wh belongs to a family of highly conserved proteins present from yeast to man. They have 5 WD40 repeats in the central portion of the protein, and a bipartite nuclear localization sequence at C-terminus. Since WD40 proteins serves as an adapter or linker in macromolecular complexes with diverse biological functions, we will be interested in investigating protein partners of wh, and uncovering the biochemical and cellular functions of wh.

Selected Publications

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