A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type φ29 DNA polymerase

摘要: Abstract Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (α-like) DNA polymerases, motif A is characterized by consensus “Dx 2 SLYP”. Mutants φ29 polymerase residue Tyr254 this had previously shown be affected dNTP binding. Here, we show that a single substitution into valine enables enzyme incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas preferred dNTPs more than two million-fold over rNTPs, mutation reduced discrimination rNTPs up 1000-fold. addition mechanism, based on sugar selection, very inefficient when extending an RNA primer terminus, allowing exonucleolytic degradation. These results indicate responsible against 2′-OH group incoming ribonucleotide. This first time invariant tyrosine involved ribo- versus deoxyribonucleotide polymerase.