Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit.
Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E.coli gamma complex clamp loader and DNA using UV-induced protein-DNA cross-linking and mass spectrometry. The results show that the delta subunit in the gamma complex makes close contact with the primer-template junction. Tryptophan 279 in the delta C-terminal domain lies near the 3'-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that delta also binds and opens the beta clamp (hydrophobic residues in the N-terminal domain of delta contact beta. The clamp-binding and DNA-binding sites on delta appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S.cerevisiae RFC complex suggests that the dual functionality observed for delta in the gamma complex may be true also for clamp loaders from other organisms.
Magdalena Coman
Maria
M
Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, CT 06459, USA.
Jin
Mi
M
Ceapa
Razvan
R
Finkelstein
Jeff
J
O'Donnell
Michael
M
Chait
Brian T
BT
Hingorani
Manju M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001973:Bromodeoxyuridine / Q000378:metabolism; D003432:Cross-Linking Reagents; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D011487:Protein Conformation; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013698:Templates, Genetic; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D021122:Protein Subunits; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D001973:Bromodeoxyuridine