Coordination between the polymerase and 5'-nuclease components of DNA polymerase I of Escherichia coli.

Abstract:

The polymerase and 5'-nuclease components of DNA polymerase I must collaborate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5'-nuclease cannot bind simultaneously to a DNA substrate and appear to compete with one another, suggesting that the two active sites are physically separate and operate independently. The desired biological end point, a ligatable nick, results from the substrate specificities of the polymerase and 5'-nuclease. The preferred substrate of the 5'-nuclease is a "double-flap" structure having a frayed base at the primer terminus overlapping the displaced strand that is to be cleaved by the 5'-nuclease. Cleavage of this structure occurs almost exclusively between the first two paired bases of the downstream strand, yielding a ligatable nick. In whole DNA polymerase I, the polymerase and 5'-nuclease activities are coupled such that the majority of molecules cleaved by the 5'-nuclease have also undergone polymerase-catalyzed addition to the primer terminus. This implies that the 5'-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution.

Polymerases:

Topics:

Nucleotide Incorporation, Exonuclease Activity, Enzyme Substrate Interactions, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow Fragment D424A Xu Y2000 3-5' Exonuclease (proofreading) No
Klenow Fragment D424A Xu Y2000 Cloned or native Cloned in E. coli
Klenow Fragment D424A Xu Y2000 5-3' Exonuclease No
Klenow Fragment D424A Xu Y2000 Template lesions Unspecified Reaction: Nucleotide incorporation
Klenow Fragment D424A Xu Y2000 Full length or truncated Full length
Klenow Fragment D424A Xu Y2000 Nick Extension Unspecified
Klenow Fragment D424A Xu Y2000 Gap Filling No
Eco Pol I D424A Xu Y2000 3-5' Exonuclease (proofreading) No
Eco Pol I D424A Xu Y2000 5-3' Exonuclease Yes
Eco Pol I D424A Xu Y2000 Full length or truncated Full length
Eco Pol I D424A Xu Y2000 Describe truncation Carrying D424A Mutation
Eco Pol I - Small Fragment Xu Y2000 3-5' Exonuclease (proofreading) Unspecified
Eco Pol I - Small Fragment Xu Y2000 Cloned or native Cloned in E. coli
Eco Pol I - Small Fragment Xu Y2000 5-3' Exonuclease Yes
Eco Pol I - Small Fragment Xu Y2000 Tagged Unspecified
Eco Pol I - Small Fragment Xu Y2000 Full length or truncated Truncated
Eco Pol I - Small Fragment Xu Y2000 Nick Extension Unspecified
Eco Pol I - Small Fragment Xu Y2000 Strand Displacement Unspecified
Eco Pol I - Small Fragment Xu Y2000 Gap Filling Unspecified
Eco Pol I - Small Fragment Xu Y2000 Describe truncation Only 5'-nuclease domain present

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