The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (APT) or incorrect (G-T, COT, T-T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon disso- energies of dissociation of correct and incorrect base pairs account for nucleotide insertion fidelity? To address these questions, a thermodynamic analysis (3) is made of melting data for oligonucleotide duplexes containing matched and mismatched template-primer termini. The thermodynamic measurements are compared with enzyme kinetic data obtained with the same DNA sequences under the same conditions, for right and wrong nucleotide insertions (4), and for elongation from matched and mismatched template-primer termini. MATERIALS AND METHODSPurified Drosophila DNA polymerase a holoenzyme (5) was a generous gift of I. R. Lehman (Stanford University, Stanford, CA). Four versions of a 20-base DNA primer (5'-TGATATTCACAACGAATGGN-3'), where N = A, C, G, or T), complementary in sequence (except for terminal base N) to bases 2242-2222 in wild-type M13 DNA (6), were synthesized by conventional solid-phase methods. The template was single-stranded DNA isolated from wild-type M13 phage grown in Escherichia coli strain JM103. Each primer was labeled at the 5' end with 32P using [y-32P]ATP (4500 Ci/ mmol; 1 Ci = 37 GBq) purchased from ICN Radiochemicals and T4 polynucleotide kinase from United States Biochemicals, Cleveland, OH. Procedures for primer 5'-end-labeling and hybridizing to template were the same as described (4).Synthetic DNA duplexes used in melting experiments, representing the last 9 base pairs in the primer-template complexes and differing only in the terminal base pair (NOT), were prepared by annealing equimolar amounts of the component 9-base strands synthesized in the same way as primers.DNA Polymerase Reactions. To measure extension rates at primer 3' ends (N opposite T), with dTTP as substrate for addition of T opposite A, reactions were carried out in the same way with each of the four 5'-end-labeled primers hybridized to M13 template as illustrated in Fig. lb

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Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.