Abstract: HU, a histone-like protein, is one of the most abundant proteins in Escherichia coli and has significant roles in DNA packaging, recombination, replication, and repair. Previous studies have shown that HU binds with high affinity, in a non-sequence specific manner, to an important intermediate in recombination and repair, known as the Holliday or 4-Way Junction. HU binds to the Holliday junction with nanomolar affinity, suggesting that recognition of specific structural elements may help to facilitate the binding between the protein and DNA. Our research aims to elucidate the structure of the HU-J20 complex to clarify the structural elements that lead to HU recognition of the Holliday Junction. In our crystallography studies, an immobile Holliday junction with 20 base pairs per strand, referred to as J20, is used to reduce the flexibility of the construct. If diffraction quality crystals are obtained, X-ray crystallography can be used to visualize structural features of the protein-DNA complex and how HU binds to J20. To develop diffraction-quality crystals, we will screen conditions that promote crystal formation. Conditions will be optimized to produce crystals that lead to high resolution diffraction patterns. The crystallographic information furthers our understanding of how HU interacts with the Holliday junction as well as its other binding partners. Additionally, by understanding how HU binds to J20, we may be able to comprehend how other proteins bind to the 4-Way Junction. Overall, the goal of this project is to solve a piece of the puzzle in HU-junction interactions in the context of recombination.
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