Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases.

By site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer-terminus at the 3'-5' exonuclease active site.     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

3'-5' exonuclease of Klenow fragment: role of amino acid residues within the single-stranded DNA binding region in exonucleolysis and duplex DNA melting

The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl-labeled primer/template indicate that the Q419A, Y423A, and R455A mutations disrupted the ability of the Klenow fragment to melt duplex DNA and bind the frayed terminus at the exonuclease site. In contrast, the N420A mutation stabilized binding of a duplex terminus to the exonuclease site, suggesting that the N420 side chain facilitates the 3'-5' exonuclease reaction by introducing strain into the bound DNA substrate. Together, these results demonstrate that protein side chains that interact with the second or third nucleotides from the terminus can participate in both the chemical step of the exonuclease reaction, by anchoring the substrate in the active site or by ensuring proper geometry of the scissile phosphate, and in the prechemical steps of double-stranded DNA hydrolysis, by facilitating duplex melting.     

Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease.

DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.     

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site.

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.     

DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization

Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel. Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II. In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity. Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity. Gel filtration indicated that the complex has a molecular mass greater than 400 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products. The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase. Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity. The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication. A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme. It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.     

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3''-5'' exonuclease and strand-displacement activities.

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.     

Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in Km values and a decrease in Vmax values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [3H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II).     

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.     

Surface structure of in vitro assembled bacteriophage lambda polyheads.

Interstrand cross-links induced by psoralen-plus-light are removed from the DNA of Escherichia coli, and this reaction is effected by the uvrA, uvrB, uvrC and polA (5′ → 3′ exonuclease) gene products. During cross-link removal, cellular DNA strands are cut so that, upon denaturation, the DNA dissociates into segments having an average molecular weight about equal to twice the average distance between cross-links. These strand cuts are persistent in cells, having a half-life of more than 20 minutes.The structure of cross-linked DNA undergoing repair was further investigated by use of density and radioactively labeled isotopes. These experiments demonstrate that two strand cuts are made in one DNA strand near each cross-link, one on each side of one arm of the cross-link. A mechanism is proposed for cross-link removal. The endonuclease coded for by the uvrA and B genes makes an incision on the 5′ side of one arm of a cross-link. Polymerase I (5′ → 3′ exonuclease) then makes a second cut on the 3′ side, in the same strand. This allows the strands to be separated during denaturation, but would leave the second arm of the cross-linking structure still attached to the uncut strand. The persistence of strand cuts at cross-links suggests that rejoining, dependent upon repair polymerization and ligation, is blocked by such a partially excised cross-linking residue. Initial stages of cross-link removal appear to be similar to pyrimidine dimer excision, but intermediates generated by these processes differ substantially in structure and repair must be completed by different mechanisms.     

Gaps in DNA induced by neocarzinostatin bear 3'- and 5'-phosphoryl termini.

Neocarzionstatin (NCS)-induced strand breakage of DNA generates nonfunctional binding sites for the E. coli DNA polymerase I. Treatment of the NCS-nicked DNA with alkaline phosphatase at 65 degrees C prior to the polymerase reaction results in 60-100-fold stimulation of dTMP incorporation whereas in a control not treated with the drug there is only a 2-fold increase. Sites of strand scission on the NCS-treated DNA bear phosphate at the 3' termini. This conclusion is supported by the kinetics of release of inorganic phosphate from NCS-cut DNA by exonuclease III. Since our earlier work has shown that virtually all the 5' ends of the nicks caused by NCS bear phosphomonoester groupings, the 3'- and 5'- phosphoryl termini could be quantitated using alkaline phosphatase and exonuclease III. Over a wide range of drug levels the amount of inorganic phosphate released by alkaline phosphatase is approximately twice as much as that removed by exonuclease III, indicating the presence of equal amounts of 3'- and 5'- phosphoryl termini. This, taken together with other previously demonstrated effects of NCS on DNA, such as the introduction of nicks not sealable by polynucleotide ligase, the release of thymine, and the formation of a malonaldehyde type compound, suggests that NCS-induced strand breakage involves base release accompanied by opening of the sugar ring with destruction of one or more nucleosides and results in a gap bounded by 3'- and 5'- phosphoryl termini.     

The 3'-5' exonuclease of human DNA polymerase delta (pol delta) is regulated by pol delta accessory factors and deoxyribonucleoside triphosphates.

Human DNA polymerase delta (pol delta) is required for the synthesis of leading strand of simian virus 40 (SV40) DNA replication in vitro. Pol delta requires the accessory factors, proliferating cell nuclear antigen (PCNA), activator 1 (A1; also known as replication factor C [RF-C]), human single-stranded DNA binding protein (HSSB; also known as replication protein A [RP-A]) for the elongation of primed template DNA. Since pol delta has an associated 3'-5' exonuclease activity, the effect of pol delta accessory factors on the exonuclease activity was examined. The 3'-5' exonuclease activity was stimulated 8-10 fold by the addition of HSSB, and this stimulatory effect was preferential to HSSB since other SSBs from E. coli, T4 or adenovirus, had a little or no effect. The stimulatory effect of HSSB was markedly inhibited by the combined action of A1 and PCNA. Furthermore, the addition of deoxyribonucleoside triphosphates (dNTPs) completely abolished the effect of HSSB on the 3'-5' exonuclease activity even in the absence of pol delta accessory factors. These results suggest that accessory factors and dNTPs regulate both the polymerase and the 3'-5' exonuclease activities.     

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.     

Mutagenic DNA repair: insertion of nucleotides opposite non-coding template structures by a reversed 3'-5' exonuclease reaction? A hypothesis.

1. The enzymatic mechanism of mutagenic DNA repair is unknown. None of the characterized DNA polymerases is capable of polymerization past non-coding template structures. 2. A hypothesis is proposed according to which polymerization opposite non-coding template structures is catalyzed by the DNA-polymerase-associated 3'-5' exonuclease under conditions which shift the equilibrium of the 3'-5' exonuclease reaction DNAn + H2O in equilibrium DNAn-1 + dNMP to the left, i.e. to the incorporation of deoxynucleoside monophosphates. 3. Conditions which favor the incorporation of dNMP by the reversed 3'-5' exonuclease reaction include a high dNMP concentration, a coupled H2O-consuming reaction and a hydrophobic enzyme environment. 4. The statements of the hypothesis are supported by published work on the biochemistry of DNA polymerases and their associated 3'-5' exonucleases, the genetics of mutagenic DNA repair and the involvement of Escherichia coli DNA polymerase III in this process. 5. The hypothesis offers an explanation of the mutator and antimutator properties of certain genes, in particular of DNA polymerase genes, and also explains how some drugs act mutagenically during DNA replication and antimutagenically against mutagenic DNA repair.     

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.     

Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, the cleavage of DNA 5' to the AP site, it displays other weak enzymatic activities. One of them is 3'-5' exonuclease activity, which is most effectively pronounced for DNA duplexes containing modified or mismatched nucleotides at the 3' end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase beta during the base excision repair process. We determined the quantitative parameters of the 3'-5' exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3' terminus of single-strand DNA and from photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Exonuclease X of Escherichia coli. A novel 3'-5' DNase and Dnaq superfamily member involved in DNA repair.

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.     

Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped DNA substrate. Biochemical analysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3'-5' exonuclease activity of PolX(Bs) resides in its PHP domain. Furthermore, site-directed mutagenesis of PolX(Bs) His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3'-termini resection by the 3'-5' exonuclease activity of PolX(Bs) in the DNA repair context are discussed.     

Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3' terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes

Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3' phosphodiesterase and 3'-5' exonuclease activities. The 3'-5' exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase beta. Here, we examine the removal of matched and mismatched dNMP from the 3' terminus of the 3'-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3' terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3' terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3'-5' exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5' margin of the nick. The 3'-5' exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5' margin of the nick as well as for flapped and recessed DNAs.