Molecular Mechanism of Type IA Topoisomerases

Authors

Tumpa Dasgupta, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

First Advisor's Name

Yuk-Ching Tse-Dinh

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Yuan Liu

Second Advisor's Committee Title

Committee Member

Third Advisor's Name

Watson Lees

Third Advisor's Committee Title

Committee Member

Fourth Advisor's Name

Prem Chapagain

Fourth Advisor's Committee Title

Committee Member

Keywords

Topoisomerases, type IA, E. coli, Mycobacterium, mechanism, domain, interaction

Date of Defense

3-23-2023

Abstract

Type IA topoisomerases relax negatively supercoiled DNA by catalyzing the breaking and rejoining of DNA single-strand coupled with DNA strand passage, thus preventing the inhibition of vital cellular processes such as transcription. The proposed mechanism predicts that the topoisomerase IA torus structure can adopt multiple open and closed conformations during the catalytic cycle. Full-length crystal structure of Escherichia coli topoisomerase I (EcTOP1) with nine domains (D1-D9) led to the hypothesis that D6 interacts with the D2-D4 hinge region through a unique D6-α1 helix to play a role in the enzyme conformational change. Two mutant EcTOP1 proteins with D6-α1 helix deleted or A651G, A655G substitution were found to have significantly reduced relaxation activity. Thermal shift assay results indicate that these α1-helix mutations most likely affect the dynamics of the opening of the torus structure. Our findings suggest that the D6 α1-helix plays a functional role in large conformational changes of EcTOP1. The proximity between D3 and D4 led to hypothesis that the perturbation of D3 and D4 interaction at the interface would make the enzyme-bridged gate opening easier or closing harder. Substitution of highly conserved E487 with alanine resulted in a mutant EcTOP1-E487A which showed 100-fold reduction in growth complementation. While the mutation does not affect the DNA binding affinity of the enzyme, EcTOP1-E487A showed decreased cleavage product formation, and reduced religation efficiency. A co-crystal of Mycobacterium tuberculosis topoisomerase I (MtbTOP1) with oligonucleotides revealed the conformational changes and the movement of several residues at the D3 and D4 interface. Based on sequence alignment, E519, E527, and R380 in MtbTOP1 were selected for alanine substitution to cause the loss of interaction between the D3-D4 interface. Experimental analysis of these mutants suggests that the interaction between E527 in D3 and residues in D4 can affect the opening of the central cavity and the folding stability of the protein but appears to be less critical than E519 for the enzyme to complete the catalytic cycle. Our findings suggest that highly conserved residues that establish the interaction of D3 and D4 of type IA topoisomerases can play a vital role in enzyme conformational changes.

Identifier

FIDC011015

Previously Published In

1. Dasgupta, T., S. Ferdous, and Y.C. Tse-Dinh, Mechanism of Type IA Topoisomerases. Molecules, 2020. 25(20).

2. Ferdous, S., et al., The interaction between transport-segment DNA and topoisomerase IA—crystal structure of MtbTOP1 in complex with both G- and T-segments. Nucleic Acids Research, 2022: p. gkac1205.

Recommended Citation

Dasgupta, Tumpa, "Molecular Mechanism of Type IA Topoisomerases" (2023). FIU Electronic Theses and Dissertations. 5308.
https://digitalcommons.fiu.edu/etd/5308