Structural and Biochemical Studies of UvrA, the Bacterial NER DNA Damage Sensor, and the Biochemical Characterization of a Bacterial MCM Protein

In this work I present a novel conformation of the Bacillus stearothermophilus UvrA (BstUvrA) protein and biochemical data to support its validity. The 2.1 A crystal structure is of a truncation of the protein lacking the UvrB-binding domain and the Insertion domain but retaining the two nucleotide binding sites. It captures a conformation in which the proximal binding site is vacant and its signature domain, signature domain II, has rotated away from the nucleotide binding site. The distal site is occupied by ADP and assumes a similar fold to that observed in the full-length BstUvrA structure. Much remains unknown about the mechanism by which UvrA deposits UvrB onto a DNA lesion to create the pre-incision complex. The rearrangement of the proximal site may represent the "disengaged" conformation of UvrA, as it leaves behind the UvrB-DNA pre-incision complex loaded at the site of DNA damage. In addition, I present work characterizing a bacterial gene product that was identified as a joint primase-MCM helicase. The novelty of this protein is that MCMs behave as the eukaryotic replicative helicase, a process which most bacteria perform with the protein DnaB. While I was able to find evidence of MCM helicase activity by the gene product, no primase activity was observed