Single‐Stranded, Primer‐Template, and Blunt‐End DNA Selectivity by Klenow and Klentaq Polymerases

Abstract

Understanding substrate selection by DNA Polymerase I is important for characterizing the balance between DNA replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the "large fragments" of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologues. We have examined the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single‐stranded DNA (ss‐DNA), primer‐template DNA (pt‐DNA), and double‐stranded DNA (ds‐DNA). The DNA binding affinity trend for Klenow from weakest to tightest binding is ds‐DNA < pt‐DNA < ss‐DNA. This is in contrast to Klentaq's DNA binding trend: ss‐DNA < pt‐DNA ≈ ds‐DNA. Both Klenow and Klentaq release more ions and have larger heat capacity changes when binding to pt‐DNA and ds‐DNA than when binding to ss‐DNA in KCl buffer. It is also found that Mg2+ significantly shifts the ds‐DNA binding affinity of Klenow, but not Klentaq. The differences in DNA structural selectivity of the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different, and that Taq polymerase is more likely to be involved in ds‐break repair and end‐preservation in vivo. Funded by the NSF and the LBRN.