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.