DNA polymerase beta: pre-steady-state kinetic analyses of dATP alpha S 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 beta from rat brain, toward Rp and Sp isomers of dATPalphaS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATPalphaS were synthesized by enzymatic methods to >98% purity. The rate of polymerization (k(pol)) and the apparent dissociation constant (K(d,app)) were measured with dATP, Rp-dATPalphaS, and Sp-dATPalphaS in the presence of Mg(2+), Mn(2+), or Cd(2+). The results indicate that wild type (WT) polymerase (Pol) beta can incorporate both Sp- and Rp-dATPalphaS in the presence of Mg(2+), but Sp is the preferred isomer. The stereoselectivity, defined as (k(pol)/K(d))(Sp)/(k(pol)/K(d))(Rp) (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg(2+). When Mg(2+) was substituted with Mn(2+) and Cd(2+), the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol beta 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 alpha-phosphate of dNTP. The kinetic data of the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPalphaS (Sp/Rp ratio = 1.5, 3.7, and 1.5 for Mg(2+), Mn(2+), and Cd(2+), respectively). Furthermore, the D276R mutant showed a 5-fold enhanced reactivity toward Rp-dATPalphaS relative to WT Pol beta, suggesting that this mutant Pol beta can be used to incorporate Rp-dNTPalphaS into DNA oligomers.
Liu
J
J
Departments of Biochemistry and Chemistry, Ohio State Biochemistry Program, and Protein Research Group, The Ohio State University, Columbus, Ohio 43210, USA.
Tsai
M D
MD
D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002104:Cadmium / Q000737:chemistry; D002413:Cations, Divalent; D004256:DNA Polymerase I / Q000737:chemistry; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics*; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D051381:Rats; D013237:Stereoisomerism; D013873:Thionucleotides / Q000138:chemical synthesis / Q000737:chemistry*
D002413:Cations, Divalent; D003838:Deoxyadenine Nucleotides; D008670:Metals; D013873:Thionucleotides; D002104:Cadmium; D001224:Aspartic Acid; D008345:Manganese; C015109:2'-deoxyadenosine 5'-O-(1-thiotriphosphate); D001120:Arginine; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D008274:Magnesium