Binding Affinity and Displacement Synthesis Activity of Pol I DNA Polymerase on Different Gapped DNAs

Abstract

Gapped DNAs, intermediates in excision repair, can be filled by DNA polymerase I (Pol I) and sealed by ligase. Understanding the binding preferences of Pol I for different gaps and how their binding affinity correlates with displacement synthesis is helpful for investigating repair mechanisms in vivo. The roles of the 5′- or 3′-phosphate, and of magnesium, in the binding of Klenow and Klentaq polymerases to gapped DNAs, differing in the size of the ssDNA gap (0, 2, and 10 nt), were examined using a fluorescence anisotropy binding assay. 5 mM Mg2+ does not significantly alter the binding of gapped DNAs to Klenow, but Mg2+ weakens the binding of gapped DNAs to Klentaq. For Klentaq, but not Klenow, a 5′-terminal phosphate increasingly weakens the binding as the gap size increases. Under the same conditions, Mg2+and 5′-phosphate do not alter the displacement synthesis ability of Klenow with gap2 and gap10 substrates, but do alter the activity of Klentaq. Conversely, a 3′-phosphate in the gap significantly weakens the binding of Klenow, but not Klentaq. We hypothesize that Klentaq can bind either the 5′- or 3′- end of the gap, while Klenow binds preferentially at 3′- end of the gap due to the 3′-end being pulled into the editing site. The binding affinity of Klenow, but not Klentaq, to different gaps increases as the size of the gap increases, and this correlates with the displacement synthesis ability of Klenow on gaps versus nicks. Klenow binds primed-template DNA substrates with 2-3 kcal/mol tighter affinity than gap0 or gap2 substrates, while Klentaq shows only a slight preference (0.7 kcal/mol) for primed-template over gapped DNAs, indicating that Klenow more significantly prefers replication over repair substrates.