Structures of normal single-stranded DNA and deoxyribo-3 '-S-phosphorothiolates bound to the 3 '-5 ' exonucleolytic active site of DNA polymerase I from Escherichia coli

The interaction of a divalent metal ion with a leaving 3' oxygen is a centr al component of several proposed mechanisms of phosphoryl transfer. In supp ort of this are recent kinetic studies showing that thiophilic metal ions ( e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this pheno menon, we have solved four crystal structures of single-stranded DNA's cont aining either oxygen or sulfur at a 3'-bridging position bound in conjuncti on with various metal ions at the 3'-5' exonucleolytic active site of the K lenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structur es of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alo ne were refined at 2.6- and 2.25-Angstrom resolution, respectively. They se rve as standards for comparison with other Mn2+- and Zn2+-containing struct ures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly id entical position to Mg2+ (Brautigam and Steitz (1998) J. Mel. Biol. 277, 36 3-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-Angstr om resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2 and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore a ppears that the ability of the bridging sulfur compounds to exclude nonthio philic metal ions from metal ion site B explains the low activity of KF exo nuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Anl. Chem. Sec. 119, 12691-12692) and that the 3'-bridging atom of the s ubstrate is influencing the binding of metal ion B prior to catalysis.