Threonine 79 Is a Hinge Residue That Governs the Fidelity of DNA Polymerase β by Helping to Position the DNA within the Active Site

Mausumi Maitra,Andrew Gudzelak,Shu-Xia Li,Yoshihiro Matsumoto,Kristin A Eckert,Joachim Jager,Joann B Sweasy

doi:10.1074/jbc.m204953200

Abstract

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.

Highlights

Mammalian dissociation constant KD (DNA) polymerase ␤1 has quickly become one of the best studied polymerases because the gene for the enzyme was cloned [1, 2]
T79S Confers a Mutator Phenotype to the SC18-12 E. coli Strain—To confirm that the candidate T79S mutant (Fig. 1) we identified in our genetic screen possessed a true mutator phenotype, we compared the spontaneous mutation frequencies of this mutant of the 8-kDa domain of pol ␤ with that of the pol ␤-wt strain using the Trpϩ reversion assay [27]
The biphasic nature of T79S is similar to WT pol ␤ [29] and indicates that the rate-limiting step of the catalytic cycle occurs after phosphodiester bond formation

Summary

Introduction

Mammalian DNA polymerase ␤ (pol ␤) has quickly become one of the best studied polymerases because the gene for the enzyme was cloned [1, 2]. It was found to interact most efficiently with the 5Ј-phosphate of the downstream primer of the gapped DNA [21, 22] This interaction is mediated by a helix-hairpin-helix motif (HhH), which is found in several other DNA repair enzymes [4, 23, 24]. The DNA polymerase active site is found in the 31-kDa domain [25]. By using our genetic screen, we isolated several mutator mutants of pol ␤, including one that is altered from Thr to Ser at position 79 (T79S). HhH motif 1 interacts with the downstream oligonucleotide, and HhH 2 interacts with the primer strand in single nucleotide gapped DNA

Results

Discussion

Conclusion