Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.
POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.
Takata
Kei-ichi
K
Department of Carcinogenesis, The University of Texas Graduate School of Biomedical Sciences at Houston, USA.
Arana
Mercedes E
ME
Seki
Mineaki
M
Kunkel
Thomas A
TA
Wood
Richard D
RD
D020816:Amino Acid Motifs; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006801:Humans; D008239:Lysine / Q000032:analysis; D009154:Mutation; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000032:analysis
D014443:Tyrosine; D004259:DNA-Directed DNA Polymerase; C546205:POLN protein, human; D008239:Lysine