Wai Mun Huang, PhD

Research Statement

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.