Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase beta by helping to position the DNA within the active site.
DNA polymerase beta (pol beta) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol beta was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol beta. Surprisingly, T79S is a misincorporation mutator only when using a 3'-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol beta. As the pol beta enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 degrees bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.
Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Mausumi
M
Maitra
Andrew
A
Gudzelak
Shu-Xia
SX
Li
Yoshihiro
Y
Matsumoto
Kristin A
KA
Eckert
Joachim
J
Jager
Joann B
JB
Sweasy
D001483:Base Sequence; D001665:Binding Sites; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry*; D016368:Frameshift Mutation; D010641:Phenotype; D013912:Threonine / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D013912:Threonine; D019951:DNA Polymerase beta