[Crystal structure of lambda-exonuclease].
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Binding Sites↗
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In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair.
Biochemical studies with model DNA heteroduplexes have implicated RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X in Escherichia coli methyl-directed mismatch correction. However, strains deficient in the four exonucleases display only a modest increase in mutation rate, raising questions concerning involvement of these activities in mismatch repair in vivo. The quadruple mutant deficient in the four exonucleases, as well as the triple mutant deficient in RecJ exonuclease, exonuclease VII, and exonuclease I, grow poorly in the presence of the base analogue 2-aminopurine, and exposure to the base analogue results in filament formation, indicative of induction of SOS DNA damage response. The growth defect and filamentation phenotypes associated with 2-aminopurine exposure are effectively suppressed by null mutations in mutH, mutL, mutS, or uvrD/mutU, which encode activities that act upstream of the four exonucleases in the mechanism for the methyl-directed reaction that has been proposed based on in vitro studies. The quadruple exonuclease mutant is also cold-sensitive, having a severe growth defect at 30 degrees C. This phenotype is suppressed by a uvrD/mutU defect, and partially suppressed by mutH, mutL, or mutS mutations. These observations confirm involvement of the four exonucleases in methyl-directed mismatch repair in vivo and suggest that the low mutability of exonuclease-deficient strains is a consequence of under recovery of mutants due to a reduction in viability and/or chromosome loss associated with activation of the mismatch repair system in the absence of RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X.
Bacterial Proteins↗
Role of the recF gene of Escherichia coli K-12 in lambda recombination.
When Escherichia coli K12(lambda) lysogens are infected with heteroimmune lambda phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which lambda replication is blocked, the recombination pathway dependent on the recF gene is fully active in producing viral recombinants even, if the phage is Red+, in the presence of exonuclease I. In contrast, removal of lambda exonuclease and beta protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitor effects of exonuclease I. In the absence of lambda exonuclease, beta protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of beta protein, full efficiency of the RecF pathway can be obtained either via cooperation with lambda exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of lambda exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that lambda exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I. When lambda replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution to the RecF pathway to lambda recombination is observed after removal of the Red system and exonuclease.
Bacteriophage lambda↗
DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions.
DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.
Alkylation↗
Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid.
Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed.
Animals↗
The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I.
The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.
Base Sequence↗
The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis.
The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.
DNA Damage↗
Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma.
The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T. A. (1985) J. Biol. Chem. 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors. Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates. The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl. In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases. Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction. In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency. The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction. To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon. In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized. As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes. Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis. It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.
Animals↗
Exonucleolytic proofreading by p53 protein.
The tumour suppressor p53 protein plays an important role in maintaining genetic integrity. Recently, p53 was shown to have an intrinsic 3'-->5' exonuclease activity. The current study has extended the characterization of purified wild-type recombinant p53-associated 3'-->5' exonuclease function to demonstrate proofreading activity. p53-associated 3'-->5' exonuclease shows clear preference for degradation of ssDNA over dsDNA substrate. On partial duplex structures, this exonucleolytic activity displays a marked preference for excision of a mismatched vs. a correctly paired 3' terminus which enables the p53 protein to act as a proofreader. However, p53 displays variation in excision of mismatched base pairs. The results demonstrate that p53 exhibits mispair excision with a specificity of A:A > A:G > A:C opposite the template adenine residue and with a specificity of G:A > G:G > G:T opposite the template guanine residue. Hence, the observed specificity of mismatch excision shows that p53 exonucleolytic proofreading preferentially repairs transversion mutations. As part of an investigation of the functional interaction between p53 and DNA polymerase, the influence of p53 on the accuracy of DNA synthesis was determined with exonuclease-deficient murine leukemia virus (MLV) reverse transcriptase (RT), representing a relatively low fidelity enzyme. Using an in vitro biochemical assay with 3'-terminal mismatch-containing DNA template primers, it was shown that wild-type recombinant p53 protein enhanced the DNA replication fidelity of MLV RT. A functional interaction between the exonuclease (p53) and polymerase (MLV RT) activities was observed; excision of mispairs by p53 was followed by further elongation onto correctly base-paired 3'-termini by MLV RT. Furthermore, the formation of 3'-mispair and subsequent mispair extension by the enzyme were decreased substantially in the presence of p53. The fact that the exonuclease-deficient MLV RT is more accurate in the presence of p53, suggests that p53 protein may function as an external proofreading exonuclease for viral enzyme. The observed decrease in initial nucleotide misincorporation and 3'-terminal mispair extension by MLV RT in the presence of p53, indicates the mechanism by which p53 affects the DNA replication fidelity of exonuclease-deficient DNA polymerase.
Baculoviridae↗
DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.
Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.
Amino Acid Sequence↗
The enzymatic basis of cyclophosphamide specificity.
Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
Animals↗
Two functional domains of the epsilon subunit of DNA polymerase III.
The epsilon subunit is the 3'-->5' proofreading exonuclease that associates with the alpha and theta subunits in the E. coli DNA polymerase III. Two fragments of the epsilon protein were prepared, and binding of these epsilon fragments with alpha and theta was investigated using gel filtration chromatography and exonuclease stimulation assays. The N-terminal fragment of epsilon, containing amino acids 2-186 (epsilon186), is a relatively protease-resistant core domain of the exonuclease. The purified recombinant epsilon186 protein catalyzes the cleavage of 3' terminal nucleotides, demonstrating that the exonuclease domain of epsilon is present in the N-terminal region of the protein. The absence of the C-terminal 57 amino acids of epsilon in the epsilon186 protein reduces the binding affinity of epsilon186 for alpha by at least 400-fold relative to the binding affinity of epsilon for alpha. In addition, stimulation of the epsilon186 exonuclease by alpha using a partial duplex DNA is about 50-fold lower than stimulation of the epsilon exonuclease by alpha. These results indicate that the C-terminal region of epsilon is required in the epsilonalpha association. To directly demonstrate that the C-terminal region of epsilon contains the alpha-association domain fusion protein, constructs containing the maltose-binding protein (MBP) and fragments of the C-terminal region of epsilon were prepared. Gel filtration analysis demonstrates that the alpha-association domain of epsilon is contained within the C-terminal 40 amino acids of epsilon. Also, the epsilon186 protein forms a tight complex with theta, demonstrating that the association of theta with epsilon is localized to the N-terminal region of epsilon. Association of epsilon186 and theta is further supported by the stimulation of the epsilon186 exonuclease in the presence of theta. These data support the concept that epsilon contains a catalytic domain located within the N-terminal region and an alpha-association domain located within the C-terminal region of the protein.
ATP-Binding Cassette Transporters↗
Dysfunctional proofreading in the Escherichia coli DNA polymerase III core.
The epsilon-subunit contains the catalytic site for the 3'-->5' proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the alpha-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the epsilon mutants were purified, and these proteins exhibited 3'-->5' exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type epsilon protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 epsilon mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified epsilon mutants with the alpha- and theta;-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active epsilon511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The epsilon511 mutant associated tightly with the alpha-subunit, but the exonuclease activity of epsilon511 was not stimulated in the alpha-epsilon511 complex. Addition of the theta;-subunit to generate the alpha-epsilon511-theta; DNA pol III core partially restored stimulation of the epsilon511 exonuclease, indicating a role for the theta;-subunit in co-ordinating the alpha-epsilon polymerase-exonuclease interaction. The alpha-epsilon511-theta; DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the alpha-epsilon511-theta; complex. Thus the epsilon511 mutant has wild-type 3'-->5' exonuclease activity and associates physically with the alpha- and theta;-subunits to generate a proofreading-defective DNA pol III enzyme.
Alleles↗
Exonucleolytic proofreading by a mammalian DNA polymerase.
Porcine liver DNA polymerase gamma contains exonuclease activity capable of digesting DNA in the 3'----5' direction, releasing deoxyribonucleoside 5'-monophosphates. The exonuclease activity excises 3'-terminal bases from both matched and mismatched primer termini, with a preference for mismatched bases. Under polymerization conditions, mismatch excision by the exonuclease occurs prior to polymerization by polymerase gamma, and this excision can be inhibited by adding to the reaction a high concentration of dNTP substrates and/or nucleoside 5'-monophosphates. In an M13mp2-based reversion assay for detecting single-base substitution errors, porcine liver polymerase gamma is highly accurate; the estimated base substitution error rate is less than one error for each 500,000 bases polymerized. Lower fidelity is observed using reaction conditions that inhibit the exonuclease activity, strongly suggesting that the exonuclease proofreads errors during polymerization. However, in a forward mutation assay capable of detecting all 12 mispairs at a variety of template positions, certain base substitution errors are readily detected even using unperturbed polymerization conditions. Thus, for some errors, polymerase gamma is not highly accurate, suggesting that proofreading is not equally active against all mispairs. To examine if the polymerase and exonuclease activities are physically as well as functionally associated, both activities were monitored during purification by four procedures, each based on a different separation principle. The two activities copurify during chromatography using phosphocellulose, heparin-agarose, or double-strand DNA-cellulose, and during velocity sedimentation in a glycerol gradient containing 0.5 M KCl. These results suggest that the polymerase and exonuclease activities are physically associated. It remains to be determined if they reside in the same subunit.
Animals↗
Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast.
The biogenesis of a number of RNA species in eukaryotic cells requires 3' processing. To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3' to 5' exonucleases. In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p). This work led to three important conclusions. First, each of these exonucleases is required for the processing of distinct RNAs. Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively. Secondly, some 3' exonucleases are redundant with other exonucleases. Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function. Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway.
Conserved Sequence↗
DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. II. Characterization of an associated endonuclease activity.
Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.
Cations, Divalent↗
Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.
The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.
Amino Acid Sequence↗
Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits.
A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.
Autoradiography↗