Structural studies of the DNA partitioning protein IncC from the plasmid RK2

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Rehman, Muhammad Fayyaz Ur (2018). Structural studies of the DNA partitioning protein IncC from the plasmid RK2. University of Birmingham. Ph.D.

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Abstract

Plasmid DNA partitioning is a crucial process for the transfer of at least a single copy of plasmid to the daughter cells during bacterial cell division. Partitioning for various low-copy number plasmids involves a DNA-binding protein (ParB), a centromere-like DNA site ( parS) and a ParA-family protein. Interestingly, the RK2 plasmid encodes two ParA proteins of different lengths. The longer protein is IncC1 (364 a.a), while IncC2 lacks a N- terminal domain of 105 amino acids (IncC NTD). The secondary structure of IncC NTD by NMR spectroscopy and other biophysical methods has been determined as random coil. It appears to bind DNA weakly and non-specifically. The expression and purification of IncC1 and IncC2 proteins was optimized. The two proteins and IncC NTD were characterized using various biophysical methods including Circular Dichroism, Analytical Ultracentrifugation, Small Angle X-ray Scattering, Size Exclusion Chromatography-Multi Angle Light Scattering, and EMSAs. Bacterial two hybrid assays and chemical crosslinking showed the two IncC proteins form homo- and hetero-dimers and interact with KorB protein. IncC1 and IncC2 proteins bind to DNA, non-specifically. IncC1 binds DNA weakly in the absence of nucleotides but IncC2 protein was found to bind DNA only in the presence of nucleotides (ADP, ATP).

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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