Spotlight on: Gary Latham

My work in the von Hippel lab focuses on understanding the assembly, maintenance, and disassembly of the T4 polymerase holoenzyme during phage replication. The T4 proteins provide an excellent reductionist model to study DNA replication, from bacteria to humans. In fact, the gp43 (polymerase), gp44/62 (clamp loader), and gp45 (sliding clamp) proteins are functionally and structurally similar to the replication components in both prokaryotic and eukaryotic systems. For example,

Replication System
Replication
factor		T4 phageE. coliHuman
Polymerasegp43 pol
Clamp loadergp44/62 or complexRF-C
Sliding clampgp45PCNA


What are roles of each of these functional components during DNA replication?

The polymerase catalyzes the transfer of nucleotide triphosphate onto the 3' -OH growing DNA primer strand. The polymerases listed above also share a 3'-5' exonuclease activity, that serves to proofread the primer terminus and ensure that exactly complementarity with the template bases is maintained.

The clamp loader has an ATPase activity that acts to couple the energy from ATP hydrolysis to loading the sliding clamp onto DNA. In all systems, the clamp loader ATPase is stimulated both by template-primer DNA and by interactions with the sliding clamp. The mechanism by which the clamp loader chaperones the ring-shaped sliding clamp onto DNA is currently unclear.

The sliding clamp is a ring-shaped protein that is known encircle the DNA like a bead on a string. For example, the structure of the protein from E. coli is known:
beta structure The dimer processity factor

As you can see, the protein is a torus, whose diameter is sufficient to accomodate double-stranded DNA. Substantial evidence in the literature supports the following model for how sliding clamps (like PCNA, , and gp45 (see above)) translocate on DNA:


The role of the sliding clamp in each system is as a processivity factor: That is, the torus encircles DNA and binds the polymerase via a protein-protein interaction. Thus the polymerase becomes tethered to the DNA by virtue of its binding to the sliding clamp, and avoids frequent dissociation from the DNA (an inefficient process that would otherwise limit the rate of DNA replication in vivo).


A image of the T4 replication fork can be viewed here.


A key question that I am working on is how does gp45 (the T4 phage sliding clamp) get loaded onto DNA? What are the structural details of the loading pathway? I have recently proposed a model, building on the previous work from the von Hippel lab and others, to describe the clamp loading cycle. This model is available to view, either as a still image or as an animated GIF.