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

Abstract

DNA polymerase ␤ (pol ␤) 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 ␤ was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol ␤ 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 ␤. 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-hairpinhelix motifs located within the 8-kDa and thumb domains of pol ␤. As the pol ␤ enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 o 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.1 has quickly become one of the best studied polymerases because the gene for the enzyme was cloned (1, 2). The availability of multiple crystal structures of human and rat pol ␤, including those of the enzyme complexed with both of its substrates and the metal cofactor, has aided the investigation of the structure-function relationships of this enzyme (3-8).pol ␤ is a 39-kDa protein with both nucleotidyltransferase and 5Ј-deoxyribose phosphodiesterase activities (9, 10). Evidence has been provided for a role for pol ␤ in both base excision repair and meiosis (11-13). There is no evidence that pol ␤ functions in replication of the mammalian genome, but pol ␤ has been shown to participate in DNA replication in Escherichia coli in the absence of DNA polymerase I (14). Mice that are completely deficient in pol ␤ die at 18 days post-conception due to massive apoptosis of post-mitotic neurons, suggesting that pol ␤ is essential for embryonic development (15, 16). The physiological DNA substrate for pol ␤ is believed to be a small gap because it has been shown that pol ␤ is processive on gaps of 6 bases or less and that the activity and fidelity of pol ␤ are highest on a 1-bp gap with a 5Ј-phosphate (17, 18).DNA polymerase ␤ has a modular organization with an 8-kDa N-terminal domain connected to the 31-kDa C-terminal domain by a protease-hypersensitive hinge region. The N-terminal 8-kDa domain was originally characterized as a singlestranded DNA binding domain (19,20). Subsequently, it was foun...

A DNA polymerase β mutant from colon cancer cells induces mutations

“…Pol ␤ lacks 3Ј to 5Ј proofreading activity, which contributes to its low fidelity. Some pol ␤ mutants can significantly increase the mutation frequency by committing errors during DNA synthesis, as documented by our laboratory (19)(20)(21), and those of others, for example (22,23). Errors committed by pol ␤ in cells may initiate or promote tumorigenesis.…”

“…The primer oligonucleotides were gel-purified as described (31) and were radiolabeled at the 5Ј end by standard methods by using T4 polynucleotide kinase (New England BioLabs) and [␥-32 P]ATP. The oligonucleotides 45A-22-22, CII45-CIIU-CIID, and CII45T-CIIU-CIIDA were annealed as described (20). The primer:template molar ratio for (CL-CP-CG) was 1.2:1:1.8 and 1.2:1:1.5 for (GL-GP-GG) in 50 mM Tris, pH 8.0, 250 mM NaCl.…”

“…We determined the equilibrium dissociation constant for dNTP binding, K d , and the maximum rate of polymerization, k pol , for correct and incorrect dNTPs for each enzyme as described (20). Single turnover conditions were determined empirically to be a ratio of enzyme to DNA of 15:1 for K289M.…”

“…The kinetics of misincorporation were determined manually under the above single-turnover conditions as described (20). For incorrect incorporation, substrate concentrations were typically 0-2 mM, and reaction times were 0-3,600 s. The product quantification and data analysis were performed as described (20).…”

“…For incorrect incorporation, substrate concentrations were typically 0-2 mM, and reaction times were 0-3,600 s. The product quantification and data analysis were performed as described (20).…”