A team led by Academician Ming-Daw Tsai, a Distinguished Research Fellow, and Dr. Wen-Jin (Winston) Wu, an Associate Research Specialist at the Institute of Biological Chemistry has discovered that human DNA polymerase μ can use a noncanonical mechanism for multiple Mn2+-mediated functions. The research article has appeared on the May 29th issue of Journal of the American Chemical Society.

Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving “prebinding” of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol μ is a unique member of the X-family with multiple functions and with unusual Mn2+ preference. In this work, this team found that Pol μ not only prebinds MdNTP in a catalytically active conformation, but also exerts the Mn2+ over Mg2+ preference at this early stage of catalysis, for various functions – incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the NHEJ repair, incorporation of dNTP to a ssDNA, and incorporation of 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn2+ over Mg2+ preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol μ . Overall, this work provides the structural and mechanistic basis for the long standing puzzle in the Mn2+ preference of Pol μ , and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways. The first author Mr. Yao-Kai Chang is in a joined PhD program between the Institute of Biochemical Sciences, National Taiwan University and the Institute of Biological Chemistry, Academia Sinica.

This research is supported by Academia Sinica, Ministry of Science and Technology (MOST) and Taiwan Protein Project. This article is the 4th major paper on DNA polymerases from Academician Tsai’s group, after the 2014 JACS paper on Africa swine fever virus Pol X, 2016 JACS paper on human Pol μ, and the 2017 review article in Nature Reviews in Chemistry.

Figure legend: In the presence of Mn2+, Pol μ follows the noncanonical reaction pathway by prebinding MndNTP, MnrNTP or Mn-8oxo-dGTP in the absence of DNA, and adopts a catalytically relevant conformation. In the presence of Mg2+, Pol μ follows the canonical pathway of DNA binding first, then MgdNTP binding. Bottom figure: Nearly identical active site structures between Pol μ:MndCTP binary complex (green), and Pol μ:DNA:MndUMPNPP precatalytic ternary complex (grey).

Article title: “Human DNA Polymerase μ Can Use a Noncanonical Mechanism for Multiple Mn2+-mediated Functions”

The full article is available at: https://pubs.acs.org/doi/10.1021/jacs.9b01741

Authors: Chang YK, Huang YP, Liu XX, Ko TP, Bessho Y, Kawano Y, Maestre-Reyna M, Wu WJ*, Tsai MD*