Ma. Trakselis et al., Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer, P NAS US, 98(15), 2001, pp. 8368-8375
Citations number
62
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (g
p45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polym
erase holoenzyme is a multistep process. A partially opened toroid-shaped g
p45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds
to this complex to generate the holoenzyme in which gp45 acts to topologica
lly link gp43 to DNA, effectively increasing the processivity of DNA replic
ation. Stopped-flow fluorescence resonance energy transfer was used to inve
stigate the opening and closing of the gp45 ring during holoenzyme assembly
. By using two site-specific mutants of gp45 along with a previously charac
terized gp45 mutant, we tracked changes in distances across the gp45 subuni
t interface through seven conformational changes associated with holoenzyme
assembly. Initially, gp45 is partially open within the plane of the ring a
t one of the three subunit interfaces. On addition of gp44/62 and ATP, this
interface of gp45 opens further in-plane through the hydrolysis of ATP. Ad
dition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformat
ion. The final holoenzyme is formed by the addition of gp43, which causes g
p45 to close further in plane, leaving the subunit interface open slightly.
This open interface of gp45 in the final holoenzyme state is proposed to i
nteract with the C-terminal tail of gp43, providing a point of contact betw
een gp45 and gp43. This study further defines the dynamic process of bacter
iophage T4 polymerase holoenzyme assembly.