Interactions of retroviral structural proteins with single-stranded nucleic acids

We have studied the interactions of single-stranded polyribonucleotides with murine leukemia virus structural proteins p10, p10' (a p10 variant), and Pr65gag, as well as Rous sarcoma virus (RSV) pp12 (a p10 analog). Two quantitative assays have been used to monitor protein-RNA association: the fluorescence enhancement of polyethenoadenylic acid) poly(epsilon A) upon binding protein, and tryptophan fluorescence quenching upon binding to poly(U). With each assay p10 was shown to bind stoichiometrically to single-stranded RNA, covering a length of nucleic acid chain (occluded site size, n) of about 6 residues. RSV pp12 was also shown to bind to poly(epsilon A), with n = 5 +/- 1. Addition of NaCl to fully titrated MuLV p10-nucleic acid mixtures effected nearly complete restoration of poly(epsilon A) or MuLV p10 fluorescence. Under conditions of 0.06 M NaCl, p10 bound noncooperatively to poly(epsilon A) with an intrinsic association constant, K = 2.3 X 10(6) M-1. K and n determined in this study were shown to relate to Kapp determined by other methods, by the approximation Kapp approximately NK, where N is the number of binding sites along the polynucleotide chain ((nucleotides/chain)/n). Chemical modifications of the p10 cysteine residues did not alter the affinity for poly(epsilon A). The affinity of Pr65gag for poly(epsilon A) appears to be higher than that of p10.     

Fluorescence studies on the complex formation between poly(rA) containing 1,N6-ethenoadenosine and poly(rU)

The interaction of the 1,N6-etheno derivatives of poly(rA) (poly(epsilon rA] with poly(rU) has been studied by absorption and fluorescence spectroscopy. The stoichiometry of the interaction is found to be 1 epsilon A:1 rU and 1 epsilon A:2 rU as well as in the case of poly(rA)-poly(rU) interaction. The fluorescence properties, including the intensity and polarization of fluorescence, respond to the conformational transition of poly(epsilon rA)-poly(rU) complexes. The introduction of epsilon A groups into poly(rA) results in a marked decrease in the melting temperature, suggesting that epsilon A may destabilize the helical structure. The three-exponential decay law obtained with poly(epsilon rA)-poly(rU) complexes indicates the existence of at least three different stacked conformational states.     

A fluorescence study of the binding of poly(1,N6-ethenoadenylic acid) to Escherichia coli initiation factor 3

The binding of initiation Factor 3 (IF3) to poly (1,N6-ethenoadenylic acid) [poly(epsilon A)] was investigated by fluorescence spectroscopy. At low salt concentrations, IF3 evokes an increase in the fluorescence intensity of poly(epsilon A) due to the unstacking of the nucleotide bases. The poly(epsilon A) fluorescence enhancement titrates to an endpoint of 13 +/- 2 nucleotide residues per IF3. The maximum poly(epsilon A) fluorescence enhancement, at lattice saturation, decreases with increasing salt concentration. Even though IF3 does not produce a large fluorescence increase between 75 and 200 mM NaCl concentration, the protein still binds to poly(epsilon A) at these salt concentrations as measured by sedimentation partition chromatography; the value of Kobs for the IF3-poly(epsilon A) interaction is comparable to that of other synthetic polynucleotides. The binding of IF3 to poly(A) at 150 and 200 mM NaCl induces an increase in nucleotide base-base separation as determined by CD, yet IF3-induced disruption of base stacking of poly(epsilon A) at these same salt concentrations is not detected by fluorescence. It is likely that IF3 binds primarily to the phosphate backbone of poly(epsilon A) at low salt concentrations, producing an increase in the fluorescence intensity. But, at higher salt concentrations, the aromatic amino acids intercalate between the nucleotide bases quenching the poly(epsilon A) fluorescence.     

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.     

Studies on the miscoding properties of 1,N6-ethenoadenine and 3,N4-ethenocytosine, DNA reaction products of vinyl chloride metabolites, during in vitro DNA synthesis.

1,N6-Ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) are formed when electrophilic vinyl chloride (VC) metabolites, chloroethylene oxide (CEO) or chloroacetaldehyde (CAA) react with adenine and cytosine residues in DNA. They were assayed for their miscoding properties in an in vitro system using Escherichia coli DNA polymerase I and synthetic templates prepared by reaction of poly(dA) and poly(dC) with increasing concentrations of CEO or CAA. Following the introduction of etheno groups, an increasing inhibition of DNA synthesis was observed. dGMP was misincorporated on CAA- or CEO-treated poly(dA) templates and dTMP was misincorporated on CAA- or CEO-treated poly(dC) templates, suggesting that epsilon A and epsilon C may miscode. The error rates augmented with the extent of reaction of CEO or CAA with the templates. Base-pairing models are proposed for the epsilon A.G. and epsilon C.T pairs. The potentially miscoding properties of epsilon A and epsilon C may explain why metabolically-activated VC and its reactive metabolites specifically induce base-pair substitution mutations in Salmonella typhimurium. Promutagenic lesions may represent one of the initial steps in VC- or CEO-induced carcinogenesis.     

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.     

Investigation of binding between recA protein and single-stranded polynucleotides with the aid of a fluorescent deoxyribonucleic acid derivative

The availability of epsilon DNA, a fluorescent ssDNA derivative, has made it possible to examine quantitatively the interactions between recA protein and single-stranded polynucleotides. Fluorescence titrations of epsilon DNA with recA protein and vice versa establish that each recA protein monomer covers 5.5 epsilon DNA nucleotides and that the dissociation constant of the recA-epsilon DNA complex is 10 nM. Fluorescence titrations of recA protein-epsilon DNA mixtures with poly(dT) establish that each recA protein monomer covers 5.1 poly(dT) nucleotides and that the dissociation constant of the recA-poly(dT) complex is 0.03 nM. Observations on how the addition of ssDNA affects the fluorescence of recA protein-epsilon DNA mixtures establish that the dissociation constant of the recA-ssDNA complex exceeds 20 microM. Stopped-flow kinetics in which excess recA protein binds to epsilon DNA indicate that k2 = 6 X 10(6) M-1 s-1 for the process. A more approximate kinetic technique indicates that recA protein binds to epsilon DNA at least one-tenth as fast as to poly(dT); the rate constant for dissociation of recA-epsilon DNA exceeds that for recA-poly(dT) by at least 30-fold. epsilon DNA is proven to be a versatile reagent for studying single-stranded polynucleotide-protein interactions. Not only can its own complexes with protein be investigated but also, under suitable circumstances, it can be used as a fluorescent probe to explore complexes incorporating nonfluorescent polynucleotides.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.     

Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study

The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.     

Fluorescence decay studies of poly(riboadenylic acid) containing 1-N6-ethenoadenosine

The fluorescence properties of the 1-N6-etheno derivatives of poly(riboadenylic acid) (poly(rA, epsilon rA)) have been examined. The fluorescence quantum yield of poly(rA, epsilon rA) decreases with an increase in the degree of the epsilon A substitution and is much smaller than that for epsilon AMP even for low degrees of epsilon A substitution. The nearest-neighbor interactions such as epsilon-adenine-adenine and epsilon-adenine-epsilon-adenine may be responsible for this behavior. It is found that the fluorescence decay kinetics obeys a three-exponential decay law for poly(rA, epsilon rA), suggesting that there exist at least three different stacked conformational states.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 2. Preparation and ribosome interaction of fluorescent Escherichia coli tRNAMetf

A fluorescent derivative of tRNAMetf from Escherichia coli has been prepared which contains 1,N6-etheno-adenosine (epsilon A) in the place of adenosine 73, the fourth residue from the 3' end. The labeled tRNA, tRNAMetf epsilon A73, is fully active with respect to aminoacylation, formylation and formylmethionyl transfer to puromycin. The preparation procedure entails the chemical removal of four nucleotides from the 3' end of tRNAMetf, ligation of the truncated molecule with epsilon A 3',5'-bisphosphate by use of T4 RNA ligase and repair of the C-C-A end with nucleotidyl transferase. The fluorescence of fMet-tRNAMetf epsilon A73 has been exploited for studying tRNA-ribosome complexes. Upon binding the tRNA into the ribosomal P site, the fluorophor experiences a change of its molecular environment as indicated by an increased fluorescence intensity. On the other hand, iodide quenching experiments indicate that, in the complex, the fluorophor is not shielded against solvent access. The results suggest that (a) adenosine 73 is not involved in direct contacts with the ribosome and (b) the stacking of the 3'-terminal A-C-C-A sequence is changed upon binding to the ribosome.     

Structural insight into poly(A) binding and catalytic mechanism of human PARN

Poly(A)-specific ribonuclease (PARN) is a processive, poly(A)-specific 3' exoribonuclease. The crystal structure of C-terminal truncated human PARN determined in two states (free and RNA-bound forms) reveals that PARNn is folded into two domains, an R3H domain and a nuclease domain similar to those of Pop2p and epsilon186. The high similarity of the active site structures of PARNn and epsilon186 suggests that they may have a similar catalytic mechanism. PARNn forms a tight homodimer, with the R3H domain of one subunit partially enclosing the active site of the other subunit and poly(A) bound in a deep cavity of its nuclease domain in a sequence-nonspecific manner. The R3H domain and, possibly, the cap-binding domain are involved in poly(A) binding but these domains alone do not appear to contribute to poly(A) specificity. Mutations disrupting dimerization abolish both the enzymatic and RNA-binding activities, suggesting that the PARN dimer is a structural and functional unit. The cap-binding domain may act in concert with the R3H domain to amplify the processivity of PARN.     

Regulation of poly(A) polymerase by 14-3-3epsilon

Poly(A) polymerase (PAP) is a key enzyme responsible for the addition of the poly(A) at the 3' end of pre-mRNA. The C-terminal region of mammalian PAP carries target sites for protein-protein interaction with the 25 kDa subunit of cleavage factor I and with splicing factors U1A and U2AF65. We used a yeast two-hybrid screen to identify 14-3-3epsilon as an additional protein binding to the C-terminal region of PAP. Interaction between PAP and 14-3-3epsilon was confirmed by both in vitro and in vivo binding assays. This interaction is dependent on PAP phosphorylation. Deletion analysis of PAP suggests that PAP contains multiple binding sites for 14-3-3epsilon. The binding of 14-3-3epsilon to PAP inhibits the polyadenylation activity of PAP in vitro, and overexpression of 14-3-3epsilon leads to a shorter poly(A) mRNA tail in vivo. In addition, the interaction between PAP and 14-3-3epsilon redistributes PAP within the cell by increasing its cytoplasmic localization. These data suggest that 14-3-3epsilon is involved in regulating both the activity and the nuclear/ cytoplasmic partitioning of PAP through the phosphorylation-dependent interaction.     

1,N6-Etheno-2-aza-adenosine 3', 5'-cyclic phosphate: human erythrocyte membrane binding and activation of membrane protein kinase.

The relative efficiency of 1,N6-etheno-2aza-adenosine 3', 5'-monophosphate (cyclic 2-aza-epsilon AMP), 1,N6-etenoadenosine 3', 5'-monophosphate (cyclic epsilon AMP) and cyclic AMP in activation of membrane protein kinase and binding to membrane was examined using isolated membranes from human erythrocytes. Cyclic 2-aza-epsilon AMP was 81% as active as cyclic AMP in erythrocyte membrane binding and activation of membrane protein kinase. On the other hand, cyclic epsilon AMP was 37% as active toward membrane protein kinase and 29% toward membrane cyclic AMP binding. Since we have previously shown that the fluorescence of cyclic 2-aza-epsilon AMP is highly sensitive to the polarity of solvents, the high efficiency of cyclic 2-aza-epsilon AMP to substitute for cyclic amp suggests that it may be a suitable microenvironmental fluorescent probe for cyclic AMP binding sites.     

Syntheses and properties of fluorescent labeled oligonucleotides containing deoxyethenoadenosine at 5' end.

The fluorescent labeled oligodeoxyribonucleotides which contain deoxyethenoadenosione (d epsilon A) at their 5' end were prepared by treating CPG bound oligonucleotides with 5'-DMTr-deoxyethenoadenosine-3'-H-phosphonate. The hybrid formation of d epsilon A-oligonucleotide with its complementary DNA was studied by fluorescence spectroscopy. The fluorescence of d epsilon A in a single strand was largely quenched by stacking interaction with the base at 3' position. When d epsilon A-oligonucleotides hybridized with their complementary strands, relative fluorescence quantum yields (Qrel) against d epsilon A changed in specific manners. These results suggest that d epsilon A-oligonucleotides are applicable to study the local structure of DNA in solution.     

A fluorescence study of the binding of eucaryotic initiation factors to messenger RNA and messenger RNA analogues

The binding of the eucaryotic polypeptide chain initiation factors (eIFs) 4A, 4B, and 4F to poly(1,N6-ethenoadenylic acid) [poly(epsilon A)] was investigated by fluorescence spectroscopy. Competition experiments allowed us to determine the relative affinity of these proteins for mRNA cap analogues and the triplets AUG, GUG, UUU, UAA, and UGA. The salt dependence of eIF-4A binding to poly(epsilon A) and mRNA suggested that the binding was largely electrostatic and was enhanced in the presence of Mg2+ and ATP. The size of the binding site of eIF-4A, eIF-4B, and eIF-4F on poly(epsilon A) was approximately 13, 25, and 35 nucleotides, respectively. Fluorescence studies with the cap analogue 7-methylguanosine triphosphate as well as competition studies with poly(epsilon A) provide further evidence for a direct interaction of eIF-4F with the cap region. There was no evidence that either eIF-4B or eIF-4A bound the mRNA cap directly. In contrast to the other two factors, eIF-4B was found to bind preferentially to AUG, and of all the triplets tested, AUG was the most effective competitor for poly(epsilon A) binding.     

5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA.

The 5'-terminal oligonucleotides m7G(5')ppp(5')NmpNp and m7G(5')ppp(5')NmpNmpNp were isolated by DEAE-cellulose column chromatography after enzymatic digestion of 32P- or methyl-3H-labeled poly(A)" HeLa cell mRNA. The recovery of such oligonucleotides indicated that a high percentage of mRNA has blocked termini. The dimethylated nucleoside, N6, O2'-dimethyladenosine (m6Am), as well as the four common 2'-O-methylribonucleosides (Gm, Am, Um, Cm) were present in the second position linked through the triphosphate bridge to 7-methylguanosine (m7G) whereas little m6Am was in the third position. The only internal methylated nucleoside, N6-methyladenosine (m6A), was found exclusively as m6ApC and Apm6ApC after digestion with RNase A, T1, and alkaline phosphatase. Digestion with RNase A and alkaline phat pyrimidines are present in much smaller amounts or absent from this position. These results imply a considerable sequence specificity since there are thousands of different mRNA species in HeLa cells. Our studies are consistent with the following model of HeLa cell mRNA in which Nm may be m6Am, Gm, Cm, Um, or Am and one or more m6A residues are present at an unspecified internal location: m7G(5')ppp(5')Nm-(Nm)---(G or A)-m6A-C---(A)100-200A.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).