PMID:10806216
| Citation |
Xu, Y, Grindley, ND and Joyce, CM (2000) Coordination between the polymerase and 5'-nuclease components of DNA polymerase I of Escherichia coli. J. Biol. Chem. 275:20949-55 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1074/jbc.M909135199 |
| Keywords |
Bacterial Proteins/metabolism; Binding Sites; DNA/metabolism; DNA Footprinting; DNA Polymerase I/metabolism; Escherichia coli/enzymology; Kinetics; Multienzyme Complexes/metabolism; Oligodeoxyribonucleotides/metabolism; Substrate Specificity |
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