Abstract
Escherichia coli
thioredoxin binds to a unique flexible loop of 71
amino acid residues, designated the thioredoxin binding domain (TBD), located
in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial
designation of thioredoxin as a processivity factor was premature. Rather it
remodels the TBD for interaction with DNA and the other replication proteins.
The binding of thioredoxin exposes a number of basic residues on the TBD that
lie over the duplex region of the primer-template and increases the
processivity of nucleotide polymerization. Two small solvent-exposed loops
(loops A and B) located within TBD electrostatically interact with the acidic
C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced
by the binding of thioredoxin. Several basic residues on the surface of
thioredoxin in the polymerase-thioredoxin complex lie in close proximity to
the TBD. One of these residues, lysine 36, is located proximal to loop A. The
substitution of glutamate for lysine has a dramatic effect on the binding of
gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on
loop B; binding is decreased 15-fold relative to that observed with wild-type
thioredoxin. This defective interaction impairs the ability of T7 DNA
polymerase-thioredoxin together with T7 helicase to mediate strand
displacement synthesis. This is the first demonstration that thioredoxin
interacts with replication proteins other than T7 DNA polymerase.