ORCID ID
Graduation Date
Summer 8-13-2021
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Programs
Pharmaceutical Sciences
First Advisor
Yuri L. Lyubchenko
Abstract
This thesis describes the nanoscale studies of protein-DNA interactions with different complexities using atomic force microscopy (AFM). One of the systems deals with DNA replication rescue. To maintain the genetic integrity, replication machinery needs to minimize the error rate, repair the damages, and restart the stalled replication caused by the attacks from the environment and inside the cell. The stalled replication rescue is orchestrated by a series of proteins, for example, the DNA helicases PriA and RecG and the ssDNA binding protein (SSB).
We demonstrated that SSB stimulates the restart process in two aspects. First, SSB facilitates the binding of PriA to the DNA substrates. Second, SSB remodels PriA, allowing the loading of PriA onto the duplex strands and the thermally driven and ATP-independent translocation of PriA. Importantly, we discovered that PriA changes the moving direction during translocation, which increases the residence time for PriA to bind to the vicinity of the replication fork. We hypothesize that PriA alters the direction by switching to the other DNA strand, a novel property of PriA that ensures the rescue at the fork position on various stalled replication forks.
Based on previous studies in the lab that revealed the remodeling of RecG by SSB, we hypothesize that the ATP-independent translocation of RecG can be stopped by the lesions in template DNA, leading to the dissociation of RecG from the DNA substrates. The results confirmed that DNA mispairings damage the binding of RecG and limit the translocation of remodeled RecG. Furthermore, the characterization of RecG dynamics on a mobile fork substrate suggests that RecG can couple the ATP-dependent fork regression with the SSB displacement during the fork rescue.
Another system focuses on the assembly of nucleosome arrays. To test the hypothesis that DNA sequence is a factor in the compaction of nucleosomes, we assembled nucleosomes on DNA substrates with different sequences. Our data showed that nucleosomes are often positioned close to each other, suggesting that non-specific sequence allows nucleosomes to communicate actively, and the internucleosomal interactions could compact nucleosomes into higher-ordered structures.
Recommended Citation
Wang, Yaqing, "Dynamics of Protein-DNA Interactions Characterized by Atomic Force Microscopy" (2021). Theses & Dissertations. 572.
https://digitalcommons.unmc.edu/etd/572