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Wai Mun Huang

professor of Pathology

B.S. Chung Chi College, Hong Kong

Ph.D. Johns Hopkins University

Research

References

waimun.huang{at}path.utah.edu

 

Research

Chromosome ends are protected and the DNA is not freely exposed.   Bacterial chromosomes and plasmids are usually circular hence they are also without free ends.   In unusual cases, an alternative conformation in the form of linear chromosomes and linear plasmids with covalent closed hairpin ends has been identified.   In these linear structures, one strand of the DNA duplex turns around and continues on to become its own complementary sequence; hence the DNA ends are again not free.   The enzymes that are responsible for generating these closed hairpin ends to allow the stable maintenance of linear chromosomes and linear plasmids are called protelomerases.   Our laboratory is interested in the structure, function and mechanism of action of this new class of protelomerases.   Thus far, protelomerases have been found mainly in gamma proteobacteria, Borrelia spirochetes and in some eukarytic brown algae viruses.   In some linear plasmids, they turn out to be the non-integrated prophages of lamda-like phages.   Protelomerases share limited amino acid sequence homology with members of tyrosine-recombinase family which use tyrosine as the active site with coordination from other conserved residues to conduct two rounds of transesterification reactions of concerted breakage and rejoining to exchange DNA strands.   In so doing, tyrosine-recombinases catalyze the insertion or removal of DNA sequences via sequence specific target sites.   Protelomerases, like topoisomerase IB and tyrosine-recombinases, also use a concerted breakage and rejoining mechanism via an active site tyrosine, but they conduct an intra-molecular reaction in a sequence specific manner to turn each of the two halves of the target site around to form two closed hair-pins ends.   We have been examining a collection of these protelomerases in a comparative study to learn the general and specific mechanistic details that govern this new class of enzymes.   We used as models the systems of linear plasmid generating systems from the more complexed phage systems of Klebsiella phage K02 and coli phage N15 to the bacterial linear chromosome generating system of Agrobacterium tumefaciens and Borrelia using genetics and biochemical means.

Another topic of continuing interest in our laboratory is in the examination of the structure and function of type II topoisomerases, the ubiquitous enzymes that are found in all living cells.   They also use a concerted breakage and rejoining mechanism to effect topological changes in DNA making them participants in various DNA transactions and are essential for cellular functions such as recombinations, transcriptions, replications and DNA segregations.   Cells frequently harbor more than one type II topoisomerases and all are essential.   In bacteria, the two type II enzymes are the homologous DNA gyrases and the topoisomerase IV's each containing two subunits.   In general, they have distinct enzymologic properties and cellular roles and the subunits are not interchangeable.   Yet there are organisms harboring only one type II enzyme indicating the one enzyme can do all type II functions in those cases. How are these unique enzymes different from the canonical gyrase and topo IV?   In small phages or viruses, host enzymes usually fulfill the roles of the needed topoisomerase functions.   More recently, new phage and viral encoded type II topoisomerases have been discovered. We are investigating under what circumstances are these new topoisomerases needed, how are they distinct from the classic enzymes of gyrase and topo IV as well as that of T4 topoisomerases.   As more atomic structures of subunits and domains of type II topoisomerases are being discovered and solved, a detailed picture and the structural rationale of what defines a gyrase and topo IV are emerging.   The comparative studies including these new topoisomerases will provide new insight into new domain organizations and functions into the general rules that govern type II topoisomerases.   Furthermore, since type II topoisomerases are targets of anti-bacterial drugs, these new topoisomerases will be valuable in applying new rational drug designs for this class of targeted drugs.

References