Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)
The fidelity of DNA synthesis by calf thymus DNA polymerase 6 (po 6) in vitro has been determined using an M13lacZa nonsense codon reversion assay. Pol 6 is highly accurate, producing on average <1 single-base substitution error for each 106 nucleotides polymerized. This accuracy is 10-and 500-fold greater than that of DNA polymerases a and (3, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol 6. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by po1 6 are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of po0 6.These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.The high base-substitution fidelity of prokaryotic DNA polymerases results from discrimination at the base insertion step and from exonucleolytic proofreading of incorrectly inserted bases during synthesis (for review, see ref.
Purified calf thymus DNA polymerases delta I and II each have an associated 3' to 5' exonuclease but otherwise resemble DNA polymerase alpha in size, biochemical kinetic parameters, and the presence of DNA primase [Crute, J. J., Wahl, A. F., & Bambara, R. A. (1986) Biochemistry 25, 26-36]. Here we demonstrate a functional association of polymerase and exonuclease with each delta form. Furthermore, we show that the exonuclease can be dissociated from DNA polymerase delta I but does not appear to be removable from DNA polymerase delta II. Polymerases delta I, delta II, and alpha are equally sensitive to the inhibitor aphidicolin, suggesting a similarity in active site structure. In comparison with DNA polymerase alpha and delta II, DNA polymerase delta I has intermediate sensitivity to 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) or N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The activity of the DNA primase of the delta II enzyme is insensitive to BuAdATP whereas 1.0 microM of this inhibitor will decrease the activity of the DNA primase of the alpha and delta I enzymes approximately 50%. Two monoclonal antibodies that potently inhibit DNA polymerase alpha are only slightly inhibitory to DNA polymerase delta I and are ineffective at inhibiting DNA polymerase delta II. DNA polymerase delta II had been previously found to be nearly inactive on nuclease-treated calf thymus DNA, relative to its activity on homopolymeric DNA. We find that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA.
The preparation, characterization and biological properties of some polyanionic polymers are reported. These polymers are constructed with repeating phenol-based monomers. The anionic groups attached to the aromatic nucleus provide the basis for binding to basic domains of proteins. Scaf-*Author to whom correspondence should be addressed.at CARLETON UNIV on June 21, 2015 jbc.sagepub.com Downloaded from 318 fold flexibility permits the polyanionic polymers to adopt low energy conformations suitable for interacting with coagulation proteins and an anti-DNA monoclonal antibody. Structure activity relationships (SAR) and comparisons with aurintricarboxylic acid are described. The polymers doubled the clotting time in the APTT assay with value ranging from 10-1000 μg/mL. Binding to an anti-DNA monoclonal antibody occurred with ICso values of 0.3-5.0 μg/mL. INTRODUCTIOÑ
The integrin receptor recognition sequence Arg-Gly-Asp was successfully used as a template from which to develop a series of potent, selective, orally active, peptide-based fibrinogen receptor antagonists with a long duration of action. Simple modifications centered on the Arg and Gly residues quickly led to a modified peptide (1) with significantly enhanced ability to inhibit in vitro platelet aggregation. Substitution of the guanidino group in 1 by piperidine provided 3, which showed not only a further increase in potency but also a modest degree of oral efficacy. Finally, exploration of the nature of the C-terminal amino acid, with respect to its side-chain functionality and the carboxy terminus, yielded a group of molecules that showed excellent in vitro potency for inhibiting platelet aggregation, excellent integrin selectivity, a high level of oral efficacy, and an extended duration of action.
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