Ultraviolet laser footprinting of histone H1(0)-four-way junction DNA complexes.

We have used a new light footprinting technique to study the interaction of histone H1(0) and a deletion mutant delta CH1(0) (lacking H1(0) COOH-terminal domain) with a synthetic four-way junction DNA. This technique is based on a single 5-ns UV laser pulse and has the ability to map protein-DNA interactions within unperturbed complexes at time scales far faster than molecular rearrangements. We found both H1(0) and delta CH1(0) to affect the photoreactivity of specific guanine residues located on the central part of four-way junction DNA. These observations demonstrate specific recognition of H1(0) for the central domain of four-way junction DNA. In addition, histone H1(0) decreases the photoreactivity of selected guanines located some distance from the crossover, indicating specific involvement of the H1(0) COOH-terminal tail with this region. Immunofractionation of delta CH1(0)-four-way DNA junction complexes with monoclonal anti-H1 antibody combined with the UV laser footprinting method demonstrated the existence of two types of delta CH1(0)-four-way DNA junction complexes.     

Role of Genetic Recombination in DNA Replication of Bacteriophage Lambda II. Effect in DNA Replication by Gene Delta

We have studied the effect of delta mutations in phage lambda on DNA synthesis as assayed by the accumulation of lambda DNA in infected cells. We find that delta mutants appear to generate somewhat less DNA than lambda(+) in a rec(+) host, suggesting the wild-type delta gene may act in DNA replication. An additional clue to delta function arises if replication is measured in the gamma-negative situation where concatemer formation is abortive. In this situation, the wild-type delta gene has an "inhibitory" effect on replication. A similar inhibitory effect on replication due to delta is observed after infection of P(2) lysogens. We conclude from these studies that the delta gene may act with alpha, beta, and gamma genes, possibly in a process affecting DNA replication.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.     

DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA

In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase delta (pol delta), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol delta is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol delta is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol delta observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a "collision release" process in which pol delta ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol delta transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol delta heterotrimer.     

T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding

To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator protein of the SV40 virus, and cognate and noncognate DNA oligomers. The results reveal two structural effects associated with "origin-specific" binding that are absent in nonspecific DNA binding. The first is the formation of a hydrogen bond (H-bond) involving His 203, a residue that genetic studies have previously identified as crucial to both specific and nonspecific DNA binding in full-length T antigen. In free T-ag-obd, the side chain of His 203 has a pK(a) value of approximately 5, titrating to the N(epsilon)(1)H tautomer at neutral pH (Sudmeier, J. L., et al. (1996) J. Magn. Reson., Ser. B 113, 236-247). In complexes with origin DNA, His 203 N(delta)(1) becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded N(delta1)H resonates at 15.9 ppm, an unusually large N-H proton chemical shift, of a magnitude previously observed only in the catalytic triad of serine proteases at low pH. The formation of this H-bond requires the middle G/C base pair of the recognition pentanucleotide, GAGGC. The second structural effect is a selective distortion of the A/T base pair characterized by a large (0.6 ppm) upfield chemical-shift change of its Watson-Crick proton, while nearby H-bonded protons remain relatively unaffected. The results indicate that T antigen, like many other DNA-binding proteins, may employ "catalytic" or "transition-state-like" interactions in binding its cognate DNA (Jen-Jacobson, L. (1997) Biopolymers 44, 153-180), which may be the solution to the well-known paradox between the relatively modest DNA-binding specificity exhibited by initiator proteins and the high specificity of initiation.     

Structural and functional properties of DNA polymerase delta from rabbit bone marrow

Summary DNA polymerase delta, the most recently described class of eukaryotic DNA polymerase, has been purified to apparent homogeneity from rabbit bone marrow. Unlike the previously known eukaryotic DNA polymerases, delta has a 3 to 5 exonuclease as an integral component of its 122 000 molecular weight, single polypeptide structure. Similar to the function with prokaryotic DNA polymerases, the 3 to 5 exonuclease assists DNA polymerase delta in maintaining the fidelity of DNA synthesis by excising misincorporated nucleotides. DNA polymerase delta and the longer known eukaryotic DNA polymerase alpha are similar in many features. Both are very sensitive to sulfhydryl inhibitors such as N-ethylmaliemide (NEM) and to the antibiotic aphidicolin. Such criteria distinguish alpha and delta from DNA polymerases beta and gamma. This has led to the conclusion that nuclear DNA replication, which is sensitive to NEM and aphidicolin, is carried out by DNA polymerase alpha. However, the similar sensitivity of delta to these reagents requires that the role of alpha and delta in nuclear DNA replication be further defined. In many features DNA polymerase delta is also similar to the viral induced DNA polymerases such as the Herpes simplex virus DNA polymerases which also have associated 3 to 5 exonuclease. Understanding of DNA synthesis and the mechanism of DNA replication fidelity in mammalian cells depends upon a further understanding of both DNA polymerases alpha and delta and the nature of the relationship they have to each other.     

Two forms of DNA polymerase delta from mouse cells. Purification and properties

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).     

1H NMR assignment and melting temperature study of cis-syn and trans-syn thymine dimer containing duplexes of d(CGTATTATGC).d(GCATAATACG)

The preparation and spectroscopic characterization of duplex decamers containing site-specific cis-syn and trans-syn thymine dimers are described. Three duplex decamers, d(CGTATTATGC).d(GCATAATACG), d(CGTAT[c,s]TATGC).d(GCATAATACG), and d(CGTAT[t,s]TATGC).d(GCATAATACG), were prepared by solid-phase phosphoramidite synthesis utilizing cis-syn and trans-syn cyclobutane thymine dimer building blocks (Taylor et al., 1987; Taylor & Brockie, 1988). NMR spectra (500 MHz 2D 1H and 202 MHz 1D 31P) were obtained in "100%" D2O at 10 degrees C, and 1D exchangeable 1H spectra were obtained in 10% D2O at 10 degrees C. 1H NMR assignments for H5, H6, H8, CH3, H1', H2', and H2" were made on the basis of standard sequential NOE assignment strategies and verified in part by DQF COSY data. Comparison of the chemical shift data suggests that the helix structure is perturbed more to the 3'-side of the cis-syn dimer and more to the 5'-side of the trans-syn dimer. Thermodynamic parameters for the helix in equilibrium coil equilibrium were obtained by two-state, all or none, analysis of the melting behavior of the duplexes. Analysis of the temperature dependence of the T5CH3 1H NMR signal gave delta H = 44 +/- 4 kcal and delta S = 132 +/- 13 eu for the trans-syn duplex. Analysis of the concentration and temperature dependence of UV spectra gave delta H = 64 +/- 6 kcal and delta S = 178 +/- 18 eu for the parent duplex and delta H = 66 +/- 7 kcal and delta S = 189 +/- 19 eu for cis-syn duplex. It was concluded that photodimerization of the dTpdT unit to give the cis-syn product causes little perturbation of the DNA whereas dimerization to give the trans-syn product causes much greater perturbation, possibly in the form of a kink or dislocation at the 5'-side of the dimer.     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.     

Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors

We employed gene targeting to study H2AX, a histone variant phosphorylated in chromatin surrounding DNA double-strand breaks. Mice deficient for both H2AX and p53 (H(delta/delta)P(-/-)) rapidly developed immature T and B lymphomas and solid tumors. Moreover, H2AX haploinsufficiency caused genomic instability in normal cells and, on a p53-deficient background, early onset of various tumors including more mature B lymphomas. Most H2AX(delta/delta)p53(-/-) or H2AX(+/delta)p53(-/-) B lineage lymphomas harbored chromosome 12 (IgH)/15 (c-myc) translocations with hallmarks of either aberrant V(D)J or class switch recombination. In contrast, H2AX(delta/delta)p53(-/-) thymic lymphomas had clonal translocations that did not involve antigen receptor loci and which likely occurred during cellular expansion. Thus, H2AX helps prevent aberrant repair of both programmed and general DNA breakage and, thereby, functions as a dosage-dependent suppressor of genomic instability and tumors in mice. Notably, H2AX maps to a cytogenetic region frequently altered in human cancers, possibly implicating similar functions in man.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

Architecture of the active DNA polymerase delta.proliferating cell nuclear antigen.template-primer complex.

The relative positions of components of the DNA-dependent DNA polymerase delta (pol delta).proliferating cell nuclear antigen (PCNA).DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol delta was nearly totally (95%) inhibited by a biotin. streptavidin complex located at the 3'-end of a template with a 15-mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin. streptavidin complex. Quantification of results with primers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol delta, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide. Interaction apparently takes place through the C terminus of p125. Based on these results, we conclude that PCNA is located "behind" pol delta in the polymerization complex during DNA synthesis and that only the large subunit of pol delta (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed.     

Interaction of Loach DNA Polymerase δ with DNA Duplexes with Single-Strand Gaps

The interaction of DNA polymerase purified from eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes with single-strand gaps of 1-13 nucleotides was studied. In the absence of template-restricting DNA, the enzyme elongated primers on single-stranded DNA templates in a distributive manner. However, in the presence of the proximal 5"-terminus restricting the template, the enzyme activity significantly increased. In this case, the enzyme was capable of processive synthesis by filling gaps of 5-9 nucleotides in DNA duplexes. These data indicate that DNA polymerase can interact with both the 3"- and 5"-termini located upstream and downstream from the gap. Analysis of the complexes formed by DNA polymerase and different DNA substrates by electrophoretic mobility shift assay confirmed the assumption that this enzyme can interact with the proximal 5"-terminus restricting the gap. DNA polymerase displayed much higher affinity in duplexes with gaps of approximately 10 nucleotides compared to the standard template–primer complexes. Maximal affinity was observed in experiments with DNA substrates containing unpaired 3"-tails in primers. The results of this study suggest that DNA polymerase exerts high activity in the cell nuclei during repair of DNA intermediates with single strand gaps and unpaired 3"-termini.     

A plasmid in the archaebacterium Methanobacterium thermoautotrophicum

The archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) was found to contain a plasmid (pME2001) in covalently closed circular form. It was isolated by CsCl gradient centrifugation of total DNA in the presence of ethidium bromide. Multimers up to the hexamer were observed upon agarose gel electrophoresis and electron microscopy of a purified plasmid preparation. A restriction map was constructed. The length of plasmid pME2001 was determined to be approximately 4,500 bp. Southern hybridization of plasmid DNA to DNA extracted from Methanobacterium thermoautotrophicum delta H (DSM1053) revealed the presence of a plasmid with homologous sequences in the delta H strain.     

A coulombic hypothesis of mitochondrial oxidative phosphorylation

A coulombic hypothesis of mitochondrial oxidative phosphorylation is presented, founded upon the evidence for negative fixed charge formation during electron transport chain activity. The intermediary force is electrostatic (psi H) and not electrochemical (delta mu H). The electrochemical potential of the chemiosmotic hypothesis is identified as a "phantom" parameter which owes its delusive existence to the procedures by which it is measured. The connection between psi H and the conditional delta mu H values is examined; it entails the use of a variable conversion factor, f, where delta mu H (mV) = f psi H, and the concept of the "protonic status" of the diffuse double layer. A number of problems which beset the chemiosmotic view are reappraised in the light of the new interpretation, and find authentic solutions.     

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).     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

The thermodynamic parameters of the conformational transitions in Mycoplasma DNA

DNA preparations have been isolated from 10 strains of phytopathogenic mycoplasms and collection culture Achole plasma laidlawii PG-8. Thermodynamic parameters of denaturation changes in the secondary structure (transconformation) of DNA of these mycoplasms have been determined. It is shown that denaturation temperature is 82.3-83.1 degree C and enthalpy of conformational DNA transitions calculated per 1 g of dry substance is 56.2-61.9 J/g. Changes in the enthalpy (delta H) and entropy (delta S) calculated per 1 mol of nucleotide pairs varied in the range of 35.6-39.0 J/m.p. and 995-109.6 J degree m.p. respectively. No linear dependence of transconformational thermal adsorption on the temperature of DNA denaturation of the studied strains of mycoplasms are revealed, which is probably connected with structural peculiarities of DNA of these microorganisms.     

Calorimetric study of aqueous solutions of histone H1 and poly-L-proline II at low temperatures

Low temperature differential scanning microcalorimetric investigation of water-histone H1 and water-poly-L-proline investigation was carried out. The concentrational dependence of the thermodynamic parameters (delta H(C), Tmax(C), delta S(T, C] for "bulk" water layers were studied. It was shown that the influence of these macromolecules on the structure and properties of surrounding water layers at the same degree of hydration is different.     

A method to select for mutator DNA polymerase deltas in Saccharomyces cerevisiae.

Proofreading DNA polymerases share common short peptide motifs that bind Mg(2+) in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase delta mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase delta mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.