A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase ␦ (pol ␦) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol ␦ on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol ␦ misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol ␦, the ability to incorporate (and/or not excise) the dTTP analog, 2-deoxythymidine-5-O-(␣-phosphonomethyl)-␤,␥-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol ␦⅐tem-plate-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol ␦. This model suggests an explicit mechanistic requirement for the intrinsic 3-5-exonuclease of pol ␦. DNA polymerase ␦ (pol ␦)1 is thought to be responsible for most DNA synthesis during mammalian replication (1). Together with its processivity factor, proliferating cell nuclear antigen (PCNA), pol ␦ is responsible for the bulk of leading strand synthesis as well as significant replication of the lagging stand. It is thought that PCNA enhances the processivity of pol ␦ by binding to both DNA and pol ␦ and by acting as a "sliding clamp" that stabilizes the interaction between pol ␦ and template-primer (2, 3). On model template-primers, PCNA stabilizes by nearly 2000-fold the pol ␦⅐template-primer interaction (4, 5) and apparently increases the rate of single nucleotide incorporation by the polymerase (6).The dramatically increased stability of the pol ␦⅐template-primer interaction in the presence of PCNA has profound functional implications. For example, it was demonstrated by O'Day et al. (7) that PCNA could facilitate synthesis by yeast pol ␦ past a thymine dimer present in the template strand. Both cis-syn and trans-syn-L-thymine dimers could be bypassed. Similarly, the ␤-subunit of Escherichia coli pol IIIholoenzyme, thought to be the functional prokaryotic homolog of eukaryotic PCNA (see Refs. 2,8), also affects replicative bypass of lesions, although in a complex way (see e.g. 9, 10). Finally, thioredoxin, the processivity subunit of T7 DNA polymerase and thus analogous to PCNA, also has effects on polymerase fidelity (11). In light of these many observations, we reasoned that addition to pol ␦ incubations of homologous PCNA might lead indirectly to an increased incidence of any relatively unlikely event (e.g. incorporation versus misincorporation).In the present study we analyzed the impact of PCNA on two different sorts of unlikely events catalyzed by pol ␦, 1)...

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Some natural and glycon-modified dNTPs with P,ypyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases a and P, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of P,y-methylenediphosphonate substituted dTTP and 3'-azido-3'-deoxy-dTTP decreased in the following order: CF 2 = CHF > CBr 2 > CFMe»CH 2 . This order is individual for each DNA polymerase. It is interesting to mention that P,Y-CBr 2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase p. This specificity distinguishes DNA polymerase P from other DNA polymerases studied.

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The reaction of phosphorylation and phosphonylation of an oligodeoxynucleotide 3'-terminal hydroxyl (oligodeoxynucleotidyl kinase activity) catalyzed by calf thymus terminal deoxynucleotidyl transferase (TDT) was found. Triphosphates modified at Palpha-, Palpha,gamma- or Palpha,beta,gamma-residues served as low-molecular weight substrates. The reaction was TDT specific; human DNA polymerasesalphaandbeta, as well as AMV reverse transcriptase did not catalyze it. The donor activity of modified triphosphates or triphosphonates depended on their structure and was increased with an increase in their hydrophobicity. The substrate activity of some modified triphosphates was up to one order of magnitude higher than that of ddTTP.

Two series of new lipophilic phosphonoformate and phosphonoacetate derivatives of AZT and d4T were synthesized and evaluated as anti-HIV agents. The efficacy of some of the synthesized compounds in cell cultures infected with HIV-1 was higher than that of the parent nucleosides and only slightly correlated to their stability in the phosphate buffer and human blood serum. The synthesized phosphonates are most probably prodrug forms of the corresponding nucleosides.

In order to develop a new generation of biocides with a wide spectrum of activities suppressing both bacteria and fungi, we have synthesized a representative library of novel 3′-modified derivatives...

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