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Mechanisms of SSB in Coordinating DNA Replication and Repair

Investigators
Linda Bloom
Institutions
University of Florida
Start date
2018
End date
2022
Objective
Mechanisms of SSB in Coordinating DNA Replication and Repair

When enzymes duplicate chromosomal DNA or repair DNA damage, the DNA duplex is unwound and regions of single-stranded (ss) DNA are exposed. Single-stranded DNA binding proteins (SSBs) play a key role in protecting DNA from damage by binding ss DNA that forms during normal cellular processes. This project addresses the questions of how SSBs bind ss DNA tightly to protect it, but at the same time allow key enzymes to access the DNA to perform their necessary activities. A combination of biochemical and biophysical techniques will be used to investigate the interactions between SSB and three different enzymes that help replicate DNA or repair DNA damage. This project will also be used as a platform for teaching scientific concepts and techniques to students of all ages in order to foster their career development. Students ranging from high school to graduate school will be actively engaged in performing the experiments. More experienced students will be encouraged to mentor newer students, thus fostering their teaching and mentoring skills, as well as solidifying their own core knowledge. The students who work on the project will also develop YouTube video vignettes that use entertaining ways to explain the science behind the project to younger students to excite young students about science and to encourage more young people to explore STEM-related career opportunities.


Single-stranded DNA binding proteins (SSBs) bind single-stranded (ss) DNA intermediates of DNA metabolism to protect ssDNA from damage and to prevent the formation of potentially mutagenic DNA secondary structures. Another critical function of SSBs is to mediate genome maintenance pathways by physically and functionally interacting with many different enzymes. These basic functions of SSB are conserved in all domains of life, but they would seem to interfere with one another. This project uses an Escherichia coli model system to uncover general mechanisms by which SSB and enzymes cooperate to enhance the efficiency of enzyme-catalyzed reactions on DNA. Despite their critical importance, mechanisms by which SSB-enzyme interactions stimulate enzyme activity and allow enzymes to gain access to DNA are not fully defined. Moreover, few studies have measured both enzyme stimulation and DNA-SSB remodeling under the same kinetic conditions to determine how the two processes are linked. This research addresses these questions by: 1) defining mechanisms by which E. coli SSB stimulates enzyme activity, and 2) defining dynamic interactions between enzymes and DNA-SSB that give enzymes access to DNA substrates. Interactions of three model enzymes, a clamp loader, a DNA polymerase, and a DNA helicase, with SSB will be investigated. These enzymes were chosen because each acts on the same DNA substrate, and thus will encounter the same dynamic DNA-SSB structure. This will increase the impact of the findings by permitting identification of mechanisms that are common to all three enzymes, and those that may be specific for a given enzyme.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
Funding Source
United States Nat'l. Science Fndn.
Project source
View this project
Project number
1817869
Categories
Escherichia coli