DNA Polymerase β: Pre-Steady-State Kinetic Analyses of dATPαS Stereoselectivity and Alteration of the Stereoselectivity by Various Metal Ions and by Site-Directed Mutagenesis
The first pre-steady-state kinetic analysis of the stereoselectivity of a DNA polymerase, Pol β from rat brain, toward Rp and Sp isomers of dATPαS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATPαS were synthesized by enzymatic methods to >98% purity. The rate of polymerization (kpol) and the apparent dissociation constant (Kd,app) were measured with dATP, Rp-dATPαS, and Sp-dATPαS in the presence of Mg2+, Mn2+, or Cd2+. The results indicate that wild type (WT) polymerase (Pol) β can incorporate both Sp- and Rp-dATPαS in the presence of Mg2+, but Sp is the preferred isomer. The stereoselectivity, defined as (kpol/Kd)Sp/(kpol/Kd)Rp (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg2+. When Mg2+ was substituted with Mn2+ and Cd2+, the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol β complexes, as well as previous steady-state kinetic studies of other DNA polymerases. In addition, the D276R mutant was designed to introduce a potential extra hydrogen bonding interaction between the arginine side chain and the pro-Sp oxygen of the α-phosphate of dNTP. The kinetic data of the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPαS (Sp/Rp ratio = 1.5, 3.7, and 1.5 for Mg2+, Mn2+, and Cd2+, respectively). Furthermore, the D276R mutant showed a 5-fold enhanced reactivity toward Rp-dATPαS relative to WT Pol β, suggesting that this mutant Pol β can be used to incorporate Rp-dNTPαS into DNA oligomers.