ATP activation of DNA polymerase III holoenzyme of Escherichia coli. I. ATP-dependent formation of an initiation complex with a primed template.

Abstract:

ATP (or dATP) stimulates DNA synthesis by DNA polymerase III holoenzyme (holoenzyme) on the synthetic template-primer poly(dA).oligo(dT)12. Nonhydrolyzable ATP analogs and other natural (deoxy)ribonucleoside triphosphates are inactive. Because the nonhydrolyzable analog 5'-deoxyadenylylimidodiphosphate is efficiently used by holoenzyme for incorporation, the ATP (or dATP) requirement for activation of replication of natural DNA could be determined. Analysis of lag times in DNA synthesis and isolation of intermediates showed that ATP (or dATP) is required in the formation of an initiation complex between holoenzyme and primed DNA template, but not for subsequent DNA synthesis. ATP is bound to holoenzyme in the absence of DNA with a KD value of 0.8 microM; 2 to 3 molecules of ATP per molecule of holoenzyme are bound without apparent cooperativity. Binding of ATP to DNA polymerase III (holoenzyme minus beta subunit) is weak (KD greater than 5 microM) and binding to the beta subunit alone is not observed. However, holoenzyme reconstituted by mixing DNA polymerase III with beta subunit binds ATP as tightly (KD = 0.6 microM) as the original holoenzyme.

Polymerases:

Topics:

Enzyme Substrate Interactions, Historical Protein Properties (MW, pI, ...), Kinetic Parameters

Note:

SSB

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Eco Pol III Holoenzyme Burgers PM1982 Molecular Weight 1.9E+06 Dalton
Eco Pol III Holoenzyme Burgers PM1982 KM 10uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Experimental conditions: ATP (100┬ÁM)

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