Structural and biochemical analysis of the UvrA binding module of the bacterial transcription repair coupling factor Mfd

The Mfd (mutation frequency decline) protein is responsible for connecting the cellular processes of transcription and DNA repair in bacteria. Mfd, also termed transcription-repair coupling factor (TRCF), recognizes arrested transcription elongation complexes and catalyzes their dissociation from damaged template DNA in an ATP-dependent manner. Subsequently, Mfd recruits the UvrABC nucleotide excision repair machinery to the damage site. The mechanistic details of this process are not fully understood. X-ray crystallography was used in order to give structural insights into the mechanism of bacterial transcription-coupled repair. During this PhD thesis, the crystal structure of the N terminus (residues 1-333) of Escherichia coli Mfd ("Mfd-N2") was solved. The Mfd N terminus is implicated to function in UvrA-binding. It bears a region with high homology to the nucleotide excision repair protein UvrB. Mfd-N2 is a triangularly shaped molecule of approximately 60×60×30 Å dimensions which contains three structural domains (domains 1A, 1B and 2). Interestingly, the structure of Mfd-N2 very much resembles that of the three N-terminal domains of UvrB. Mfd domain 1A adopts a typical RecA fold. However, it lacks the functional motifs of active ATPases, and we could confirm that the Mfd N-terminus does not possess any ATPase activity. Domain 1B matches the damage-binding domain of the UvrB. Interestingly, Mfd is bereft of the damage-binding motif of UvrB domain 1B, and no DNA binding is associated with this part of Mfd. Domain 2, which possesses the highest sequence homology to UvrB, closely matches the three-dimensional structure of the implicated UvrA-binding domain of UvrB. Highly conserved amino acids between Mfd and UvrB can be found on the surface of domain 2. Using site-directed mutagenesis, several of these residues could be shown to function in the UvrA-Mfd interaction. Remarkably, the corresponding residues in UvrB are required for productive interaction between UvrA and UvrB as well. Taken together, these results suggest that Mfd and UvrB interact with UvrA in a similar manner. Mfd may form an UvrA-recruitment factor at stalled transcription complexes that resembles UvrB architecturally but not catalytically. The molecular similarity between Mfd and UvrB indicates an evolutionary connection between global genome and transcription-coupled nucleotide excision repair in bacteria