Browsing by Author "Hsieh, Tao-shih"

Eukaryotic Topoisomerase 2 is an essential enzyme that solves DNA topological problems such as DNA knotting, catenation, and supercoiling. It alters the DNA topology by introducing transient double strand break in one DNA duplex as a gate for the passage of another DNA duplex. Two different aspects of studies about eukaryotic Topoisomerase 2 will be covered in this thesis. In the first half of the thesis, we investigated conformational changes of human Topoisomerase 2 (hsTop2) in the presence of cofactors and inhibitors. In the second half, we focused on an unknown regulatory function in the C-terminal domain (CTD) of Drosophila Topoisomerase 2 (Top2).

In the project of studying enzyme conformational changes, we adapted a previously developed methodology, Pulse-Alkylation Mass Spectrometry, with monobromobimane to study the protein dynamics of hsTop2. Using this method, we captured the evidence of conformational changes in the presence of ATP and Mg2+ or the Top2 inhibitor, ICRF-193 which were not previously observed. Last, by using CTD truncated hsTop2, the increasing reactivity of Cys427 suggested the CTD domain might be tethered adjacent to the core enzyme.

Following the study of enzyme conformational changes, we switched gear to examine an interaction between Drosophila Top2 and Mus101, homolog of human TopBP1. We first found that Mus101 interacts with CTD of Top2 in a phosphorylation-dependent manner. Next, in the co-immunoprecipitation and pull-down experiments using truncated or mutant Top2 with various Ser to Ala substitutions, we mapped the binding motif to the last amino acids of Top2 and identified that phosphorylation of Ser1428 and Ser1443 is important for Top2 to interact with the N-terminus of Mus101, which contains BRCT1/2 domains (BRCT, BRCA1 C-terminus). The binding affinity of the N-terminal Mus101 with a synthetic phosphorylated peptide covering the last 25 amino acids of Top2 (with pS1428 and pS1443) was determined by surface plasmon resonance with a Kd of 0.57 μM. In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response to Mus101. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top220, truncation of 20 amino acids from the C-terminus of Top2, developed abnormally high chromosome numbers, which implies an infidelity in chromosome segregation during mitosis. Lastly, Top2-null flies rescued by Top2 with S1428A and S1443A were found to be viable but sterile. After investigating spermatogenesis, telophase of meiosis I was delayed, indicating Top2-Mus101 interaction is also important in segregating DNA in meiosis.

RecQ4, a member of the conserved RecQ family of helicases, is involved in replication and associated with several clinical syndromes. Although biologically important, the biochemistry of RecQ4 has remained elusive. We have expressed and purified Drosophila melanogaster RecQ4 from a baculovirus expression system. Biochemical characterization of the helicase, ATP hydrolysis, annealing, and binding activities of the enzyme has been performed, using native and non-native gel electrophoresis and thin layer chromatography, among other techniques. These reveal that RecQ4 is a 3' to 5' helicase that is stimulated by the presence of single-stranded DNA 3' of the duplex DNA region to be unwound. The enzyme is also capable of annealing complementary DNA strands, though this is inhibited by AMPPNP, a non-hydrolyzable analog of ATP. RecQ4 also forms a stable complex with single-stranded DNA in the presence of AMPPNP. We argue that the helicase activity of RecQ4 is important to the process of DNA replication. This leads to the conclusion that two helicases, RecQ4 and the Mcm2-7 complex, are involved in replication. The manner of their simultaneous involvement is not intuitive, and so models by which the two enzymes may cooperate are discussed.