The kinetic mechanism of formation of the bacteriophage T4 DNA polymerase sliding clamp

Journal of Molecular Biology
M C YoungP H von Hippel

Abstract

DNA replication in bacteriophage T4 requires the assembly of a structure called the "sliding clamp" near the 3' end of the DNA strand that is to be extended. This structure is a trimer ring of the T4 gene 45 product (gp45) and serves to regulate the processivity of the DNA polymerase within the T4 DNA replication system. The placement of this ring is performed by an ATPase complex of the products of T4 genes 44 and 62 (gp44/62) that consists of four gp44 subunits and one gp62 subunit. In an effort to understand the role of ATP hydrolysis in processes occurring during the formation of the phage T4 DNA sliding clamp, we have performed direct substrate and product binding experiments and steady-state and presteady kinetic experiments on the gp44/62-gp45 system. Substrate (ATP) and product (ADP) binding studies show that the gp44/62 complex binds 4(+/-1) ATP molecules with a Kd of 34(+/-12) microM, and 3.7(+/-0.3) ADP molecules with a Kd of 14(+/-7) microM. The binding of the other reaction product (inorganic orthophosphate) could not be detected. Presteady-state kinetic analysis of ATP hydrolysis during the sliding-clamp-loading process indicates a biphasic progress curve, consisting of an initial rapid "burst" phase with an ampli...Continue Reading

Citations

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