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

The polymerase and 5'-nuclease components of DNA polymerase I must collabor ate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5'-nuclease cannot bind simultaneously to a DNA substra te and appear to compete with one another, suggesting that the two active s ites are physically separate and operate independently, The desired biologi cal end point, a ligatable nick, results from the substrate specificities o f the polymerase and 5'-nuclease. The preferred substrate of the 5'-nucleas e 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 suc h that the majority of molecules cleaved by the 5'-nuclease have also under gone polymerase-catalyzed addition to the primer terminus. This implies tha t the 5'-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution.