Hydrophobic interactions in the hinge domain of DNA polymerase beta are important but not sufficient for maintaining fidelity of DNA synthesis.
We previously described a general mutator form of mammalian DNA polymerase beta containing a cysteine substitution for tyrosine 265. Residue 265 localizes to a hydrophobic hinge region predicted to mediate a polymerase conformational change that may aid in nucleotide selectivity. In this study we tested the hypothesis that van der Waals and hydrophobic contacts between Y265 and neighboring residues are important for DNA synthesis fidelity and catalysis, by altering interactions in the hinge domain via substitution at position 265. Consistent with the importance of hydrophobic interactions, we found that phenylalanine, leucine, and tryptophan substitutions did not alter significantly the steady-state catalytic efficiency of DNA synthesis, relative to wild type, while the polar serine substitution decreased catalytic efficiency 6-fold. However, we found that all substitutions other than phenylalanine increased the error frequency, relative to wild type, in the order serine > tryptophan = leucine. Therefore, maintenance of the hydrophobicity of residue 265 was not sufficient for maintaining fidelity of DNA synthesis. We conclude that while hydrophobic interactions in the hinge domain are important for fidelity, additional factors such as electrostatic and van der Waals interactions contributed by the tyrosine 265 aromatic ring are required to retain wild-type fidelity.
Opresko
P L
PL
Department of Biochemistry and Molecular Biology and The Jake Gittlen Cancer Research Institute, Pennsylvania State University College of Medicine, M. S. Hershey Medical Center, P.O. Box 850, Hershey, Pennsylvania 17033, USA.
Shiman
R
R
Eckert
K A
KA
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D004252:DNA Mutational Analysis; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication / Q000235:genetics; D016368:Frameshift Mutation; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D014764:Viral Proteins; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D013937:Thymidine Kinase; D019951:DNA Polymerase beta; D007930:Leucine