Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy.

[摘要] The error-free progression of DNA replication is essential for all organisms. Approximately 80 known human diseases are caused by malfunction in DNA replication, and deficiencies in DNA repair mechanisms can also lead to severe consequences such as increased antibiotic resistance in bacteria and cancers in humans. A better understanding of DNA replication and repair requires knowledge of the key players along relevant pathways at the molecular level and in the cellular context. This characterization calls for a technique with superior sensitivity, accuracy and biocompatibility. In this thesis, we integrate single-molecule super-resolution microscopy and single-particle tracking with genetic and genomic approaches to study two proteins that play a pivotal role in maintaining genomic integrity: MutS and PolC. From prokaryotes to human cells, homologs of the highly conserved mismatch repair (MMR) protein MutS recognize mispaired nucleotides and recruit the proteins responsible for downstream repair. Although the structure and function of MutS have been extensively characterized in biochemical isolation, it remains unclear how MutS efficiently identifies, among millions of correctly paired bases, a single mismatch in the complex and crowded cellular environment. To obtain mechanistic insights into MMR initiation from an in vivo perspective, we applied super-resolution imaging in live Bacillus subtilis cells to follow the motion of single MutS proteins in real time, and we monitored how MutS behavior is affected by sequentially blocking critical steps along the MMR pathway. Our results demonstrate an intimate and dynamic coupling between MutS and the replisome which stages MutS to sites of DNA replication, allowing MutS to scan newly synthesized DNA in anticipation of errors largely free of obstacles.We then turn our focus to DNA replication itself. Specifically, we focused on PolC, one of the two essential DNA polymerases in B. subtilis. Based on photobleaching-assisted microscopy and three-dimensional super-resolution microscopy, we quantified the stoichiometry, intracellular locations and dynamics of PolC.Finally, we extended the application of super-resolution microscopy to the field of renewable energy by tracking single molecules and visualizing guest-host interactions in microporous coordination polymers (MCPs).

[发布机构] University of Michigan

[效力级别] Single-molecule imaging

[关键词] Super-resolution microscopy;Single-molecule imaging;DNA mismatch repair;Chemistry;Science;Chemistry