Human DNA polymerase λ diverged in evolution from DNA polymerase β toward specific Mn⁺⁺ dependence: A kinetic and thermodynamic study

The recently discovered human DNA polymerase λ (DNA pol λ) has been implicated in translesion DNA synthesis across abasic sites. One remarkable feature of this enzyme is its preference for Mn²⁺ over Mg²⁺ as the activating metal ion, but the molecular basis for this preference is not known. Here, we present a kinetic and thermodynamic analysis of the DNA polymerase reaction catalyzed by full length human DNA pol λ, showing that Mn²⁺ favors specifically the catalytic step of nucleotide incorporation. Besides acting as a poor coactivator for catalysis, Mg²⁺ appeared to bind also to an allosteric site, resulting in the inhibition of the synthetic activity of DNA pol λ and in an increased sensitivity to end product (pyrophosphate) inhibition. Comparison with the closely related enzyme human DNA pol β, as well as with other DNA synthesising enzymes (mammalian DNA pol α and DNA pol δ, Escherichia coli DNA pol I, and HIV-1 reverse transcriptase) indicated that these features are unique to DNA pol λ. A deletion mutant of DNA pol λ, which contained the highly conserved catalytic core only representing the C-terminal half of the protein, showed biochemical properties comparable to the full length enzyme but clearly different from the close homologue DNA pol β, highlighting the existence of important differences between DNA pol λ and DNA pol β, despite a high degree of sequence similarity.