Transcription-Coupled DNA Repair in Prokaryotes - Progress in Molecular Biology - page 31

Interaction between Mfd and RNA polymerase is crucial toECdisplacement,
and it involves the RNA polymerase interaction domain (RID), D4, of Mfd and
the subunit of the polymerase. Studies of the effects of mutations in these

regions, together with structural studies of the individual proteins and of the
Mfd -RID complexed with the

b

subunit,
have provided information about the nature of the interaction and the amino
acids involved. For example, a mutation in D4, L499R, abolishes the displace-
ment of RNA polymerase from DNA but not the DNA binding, ATP binding,
or ATP hydrolysis by Mfd . 28 In many of the structural studies, the Mfd protein
analyzed has been in the closed conformation in which the enzymatic activities,
including RNA polymerase displacement, are inhibited. 17,28,32,36 What deter-
mines the conformation of the protein in vivo , and exactly how the Mfd protein
initiates a productive interaction with the EC that results in the forward
movement or the dissociation of the RNA polymerase from DNA remains
puzzling.
In contrast to Mfd , when an EC encounters another EC blocked by a
protein bound to the DNA, the upstream EC may help the downstream EC
overcome the block. Two upstream ECs to ''push'' the downstream EC past the
block are more efficient than one. Unlike Mfd , the upstream EC does not
displace the downstream EC but allows it to continue transcription. 41,42 What
determines the outcome of such encounters is not understood.
In addition to the RID, the superfamily 2 translocation module (D5 and
D6) of Mfd is required for the displacement of a stalled or blocked EC. Some
sequences of this ''motor domain'' are homologous to sequences in the RecG
protein, and like RecG, Mfd is an ATP-dependent double-strand DNA translo-
case. Certain substitutions in the TRG domain (e.g., R953A and Q963A)
abrogate RNA polymerase displacement in vitro and in vivo without interfer-
ing with DNA binding and nucleotide hydrolysis. 28,37
In addition to displacing the EC blocked at a lesion, Mfd recruits UvrA .
The UvrB homology module (D1a, D2, D1b) of Mfd is absolutely required for
this, 39 but the details of the mechanism involved have not been elucidated. In
the case of GGR, Orren and Sancar 43 and Lin and Sancar 44 proposed that a
heterotrimer composed of two UvrA molecules and one UvrB molecule initi-
ated the identification of a lesion. Subsequently, Malta et al. 45 presented
evidence that the identification involved a heterotetramer of two UvrA mole-
cules and two UvrB molecules, with the two UvrB molecules then being loaded
onto the DNA by UvrA . More recently, using Q-dot technology, Kad et al. 46
confirmed that the UvrA 2 UvrB 2 heterotetramer could form even in the ab-
sence of DNA, but found that it was not necessary for loading UvrB on DNA.
Although UvrB did not bind to DNA in the absence of UvrA , the UvrA
homodimer did bind to DNA in the absence of UvrB, and UvrB could then
associate with the bound UvrA . Under the experimental conditions used, UvrB

b 1 portion of the RNA polymerase b