An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a 'steric gate' residue for discrimination against ribonucleotides.
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.
DeLucia
Angela M
AM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Grindley
Nigel D F
ND
Joyce
Catherine M
CM
D019843:Archaeal Proteins; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D016368:Frameshift Mutation; D007700:Kinetics; D016254:Mutagenesis, Insertional; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D016944:Sulfolobus / Q000201:enzymology
D019843:Archaeal Proteins; D003854:Deoxyribonucleotides; D009841:Oligonucleotides; D012265:Ribonucleotides; C436950:Dbh protein, Sulfolobus solfataricus; D004259:DNA-Directed DNA Polymerase