Nucleic Acids Research, 2003, Vol. 31, No. 14 4129-4137
© 2003 Oxford University Press
An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a ‘steric gate’ residue for discrimination against ribonucleotides
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
*To whom correspondence should be addressed at Department of Molecular Biophysics and Biochemistry, Yale University, Bass Center for Molecular and Structural Biology, 266 Whitney Avenue, PO Box 208114, New Haven, CT 06520-8114, USA. Tel: +1 203 432 8992; Fax: +1 203 432 3104; Email: catherine.joyce{at}yale.edu
DNA polymerases of the A and B families, and reverse transcriptases, share a common mechanism for preventing incorporation of ribonucleotides: a highly conserved active site residue obstructing the position that would be occupied by a 2' hydroxyl group on the incoming nucleotide. In the family Y (lesion bypass) polymerases, the enzyme active site is more open, with fewer contacts to the DNA and nucleotide substrates. Nevertheless, ribonucleotide discrimination by the DinB homolog (Dbh) DNA polymerase of Sulfolobus solfataricus is as stringent as in other polymerases. A highly conserved aromatic residue (Phe12 in Dbh) occupies a position analogous to the residues responsible for excluding ribonucleotides in other DNA polymerases. The F12A mutant of Dbh incorporates ribonucleoside triphosphates almost as efficiently as deoxyribonucleoside triphosphates, and, unlike analogous mutants in other polymerase families, shows no barrier to adding multiple ribonucleotides, suggesting that Dbh can readily accommodate a DNA–RNA duplex product. Like other members of the DinB group of bypass polymerases, Dbh makes single-base deletion errors at high frequency in particular sequence contexts. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis, indicating that the geometry of nucleotide binding is similar in both circumstances.
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