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• W2130629674 endingPage "452" @default.
• W2130629674 startingPage "440" @default.
• W2130629674 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 aKdof 34(±12) μM, and 3.7(±0.3) ADP molecules with aKdof 14(±7) μM. 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 amplitude of four ATP molecules per gp44/62 complex and a rate of 15 s−1, followed by a second slower phase corresponding to the steady-state rate of ATP hydrolysis by this complex. The rate of the burst phase is kinetically consistent with the previously observed rate of T4 DNA polymerase holoenzyme formation. The burst amplitude depends solely on the concentration of gp44/62 ATP binding sites present. These results suggest that the formation of a single T4 sliding clamp requires the hydrolysis of four ATP molecules by one gp44/62 complex in a process requiring 0.5 to 1 second. A model describing the clamp-loading process is discussed in the context of these results." @default.
• W2130629674 created "2016-06-24" @default.
• W2130629674 creator A5035447333 @default.
• W2130629674 creator A5057651153 @default.
• W2130629674 creator A5070130253 @default.
• W2130629674 date "1996-12-01" @default.
• W2130629674 modified "2023-09-26" @default.
• W2130629674 title "The Kinetic Mechanism of Formation of the Bacteriophage T4 DNA Polymerase Sliding Clamp" @default.
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• W2130629674 doi "https://doi.org/10.1006/jmbi.1996.0652" @default.
• W2130629674 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8969296" @default.
• W2130629674 hasPublicationYear "1996" @default.
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