Sc. Alley et al., Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly, BIOCHEM, 39(11), 2000, pp. 3076-3090
The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polym
erase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is
loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring
-shaped gp45 is loaded onto DNA such that the DNA passes through the center
of the ring. gp43 binds to this complex, thereby forming a topological lin
k with the DNA and increasing its processivity. Using stopped-flow fluoresc
ence-resonance energy transfer, we have investigated opening and closing of
the gp45 ring during the holoenzyme assembly process. Two amino acids that
lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled
with coumarin, were used as the fluorescence donor and acceptor, respectiv
ely Free in solution, gp45 has two closed subunit interfaces with W91 to V1
62-coumarin distances of 19 Angstrom and one open subunit interface with a
W91 to V162C-coumarin distance of 40 Angstrom. Making the assumption that t
he distance across the two closed subunit. interfaces is unchanged during t
he holoenzyme assembly process, we have found that the distance across the
open subunit interface is first increased to greater than 45 Angstrom and i
s then decreased to 30 Angstrom during a 10-step assembly mechanism. The gp
45 ring is not completely closed in the holoenzyme complex, consistent with
previous evidence suggesting that the C-terminus of gp43 is inserted into
the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled
to only a fraction of the total distance change, with conformational chang
es linked to binding DNA and gp43 coupled to the majority of the total dist
ance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in
formation of a nonproductive gp45 gp44/62 complex; however, adding an exces
s of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exc
hange to yield a productive gp45 gp44/62 complex within seconds.