Cytidine Triphosphate Specifically Modulates ParB-Mediated DNA Compaction: Insights From Single-Molecule Analysis and ParB C-Terminal Lysine Mutagenesis

Author

Lois Akosua Serwaa, The University of Texas Rio Grande Valley

Date of Award

7-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

First Advisor

HyeongJun Kim

Second Advisor

Nirakar Sahoo

Third Advisor

Yonghong Zhang

Abstract

ParB is a central player in bacterial chromosome segregation, and cytidine triphosphate (CTP) is known to modulate its DNA compaction activity. This study explored whether CTP’s regulatory effect is due to specific molecular interactions beyond its known CTP-binding pocket. Using single-molecule flow-stretching and TIRF microscopy, we showed that CTP significantly reduces DNA compaction by wild-type Bacillus subtilis ParB, while ATP has no effect, supporting the hypothesis that CTP acts through specific recognition rather than general charge. CTP’s modulatory effect persisted across C-terminal domain (CTD) lysine mutants. Compaction was reduced in wild-type and K255A but abolished in K252A and K259A. The triple mutant (K252A-K255A-K259A) showed slight CTP-enhanced activity, suggesting that CTP’s influence may extend beyond its binding pocket. These findings support the hypothesis that negatively charged CTP modulates ParB's DNA compaction through both the CTP-binding pocket and DNA-binding regions, offering new insight into how nucleotide specificity governs ParB-DNA interactions.

Comments

Copyright 2025 Lois Akosua Serwaa. All Rights Reserved. https://proquest.com/docview/3254036758

Recommended Citation

Serwaa, L. A. (2025). Cytidine triphosphate specifically modulates ParB-mediated DNA compaction: Insights from single-molecule analysis and ParB C-terminal lysine mutagenesis [Master's thesis, The University of Texas Rio Grande Valley]. ScholarWorks @ UTRGV. https://scholarworks.utrgv.edu/etd/1763