The DNA polymerases that are specialized for chromosomal replication operate with exceptional speed and fidelity. This highly processive movement along the DNA template is enabled by the utilization of a specialized system of sliding clamps and ATP-dependent clamp loader complexes that load the sliding clamps on to DNA. The first major discovery from our group in this area was the determination of the structures of the sliding clamps of E. coli and yeast, which established the now general principle that sliding clamps of chromosomal replicases are circular proteins that provides a mobile tether on DNA (Kong et al., Cell 1992
; Krishna et al., Cell 1994
). This was followed by the determination of the structures of the clamp loader complexes from E. coli and yeast (Bowman et al., Nature 2004)
. The clamp loaders are part of a very large and diverse family of oligomeric ATPases known as AAA+ ATPases and, along with work from other groups on AAA+ proteins involved in vesicle fusion, the structures from our lab, in collaboration with Mike O’Donnell
, provided the first view of the architecture of these proteins.
Clamp loaders recognize a special DNA structure known as a primer-template junction. By analyzing structures of clamp loaders loaded with ATP analogs and bound to closed and open clamps and to primer-template junctions, we explained how the clamp loader recognizes the primed DNA specifically, and how the recognition of DNA is coupled to ATP hydrolysis and release of the closed clamp on DNA (Kelch et al., Science 2011)
Bacterial chromosomal DNA replicases, such as E. coli DNA polymerase III (Pol III) share no sequence similarity with other DNA polymerases, including the eukaryotic DNA polymerases that replicate chromosomes. We determined the crystal structure of a large (~900 residue) fragment of E. coli DNA polymerase III (Lamers et al., Cell 2006)
, which reveals a unique chain fold with localized similarity at the active site to DNA polymerase β, a member of the nucleotidyl transferase family. The structure of Pol III bears no similarity to that of canonical DNA polymerases, including the chromosomal replicases of eukaryotes and archae, suggesting that the DNA replication machinery arose independently in bacteria. The structure suggests a model for how the catalytic subunit of Pol III engages the sliding clamp and DNA. Generally similar conclusions were drawn by Thomas Steitz
and colleagues, who independently determined the structure of the DNA polymerase from Thermus acquaticus.