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PubMed2000

Selective blockage of the 3'-->5' exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA.

Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3'-->5' helicase and 3'-->5' exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo-tides. In addition, we directly compared the 3'-->5' WRNp exonuclease activity with that of exo-nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo-nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo-tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.

Adenine↗
PubMed2000

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA.

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3'-->5' DNA helicase and associated ATPase activities, as well as 3'-->5' DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.

Adenosine Triphosphatases↗
PubMed2006

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance.

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.

DNA↗
PubMed1979

Role of exonucleases V and VIII in adenosine 5'-triphosphate- and deoxynucleotide triphosphate-dependent strand break repair in toluenized Escherichia coli cells treated with X-rays.

The repair of X-ray-induced strand breaks was studied in permeabilized Escherichia coli recBC cells deficient for the adenosine 5'-triphosphate (ATP)-dependent exonuclease V and in recBC sbcA cells that possess the ATP-independent exonuclease VIII. It is shown that repair induced by additon of ATP does not take place in recBC and recBC sbcB cells and is limited in recBC sbcA cells. ATP-dependent repair is nevertheless observable if together with ATP a mixture of deoxynucleotide monophosphates is supplied to the cells. These data fit with the assumption that in wild-type cells ATP-dependent repair involves exonuclease V-induced deoxyribonucleic acid degradation and rephosphorylation of the degradation products which are reused for deoxyribonucleic acid polymerase I-dependent break closure. Repair in the presence of deoxynucleotide triphosphates rejoins a similar fraction of breaks in all strains tested irrespective of the amount of postirradiation degradation resulting from exonuclease V and exonuclease VIII activities. Thus, exonuclease V is dispensable for deoxynucleotide triphosphate-dependent repair, i.e., does not "clean" the ends of breaks produced by X-irradiation. ATP- and deoxynucleotide triphosphate-dependent repair are not additive and seem to repair the same population of deoxyribonucleic acid molecules damaged by X-irradiation.

Adenosine Triphosphate↗
PubMed1974

T7 exonuclease (gene 6) is necessary for molecular recombination of bacteriophage T7.

The role of T7-induced exonuclease (gene 6) in molecular recombination was studied by examining the fate of parental DNA during parental-to-progeny recombination. The method used was to compare infections with T7(+), T7am-6-233 (am gene 6), or T7ts6-136 (ts gene 6) under permissive and nonpermissive conditions. CsCl density gradient analysis of replicative DNA indicated that T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Further studies under conditions where replicative DNA was denatured and analyzed by CsCl density gradient centrifugation indicated that the exonuclease is also needed for a limited amount of covalent repair of recombinants. A repair function for the T7-induced exonuclease is also suggested by results obtained from alkaline sucrose gradient analysis of replicative DNA. Under conditions nonpermissive for the exonuclease, discontinuities in the DNA accumulated during infection by T7am6-233 or by T7ts6-136.

Bromodeoxyuridine↗
PubMed1982

mRNA- and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes.

Microinjection of herpes simplex virus (HSV)-infected cell mRNA into Xenopus laevis oocytes resulted in the production of a new exonuclease activity. This enzyme strongly resembled the HSV alkaline exonuclease in many biochemical properties, and hybrid-arrested translation studies showed that it was virus coded, mapping at 0.080 to 0.185 genome map units. Exonuclease mRNA had a size and genome location equivalent to the mRNA encoding V185 in reticulocyte lysates, suggesting that V185 is the exonuclease. The enzyme synthesized in oocytes was found to act as an exonuclease in vivo. Two plasmids containing HSV DNA fragments directed the synthesis of exonuclease when microinjected into oocyte nuclei, and this finding enabled the coding and control sequences for this gene to be localized to 0.155 to 0.185 genome map units.

Animals↗
PubMedDate not supplied

[Complexes of nuclear DNA-polymerases with 3'----5'-exonucleases from the rat liver].

"Editing" 3'----5' exonuclease activity of DNA polymerases corrects replication errors. This activity associated with procaryotic DNA polymerases is not intrinsic to purified mammalian DNA polymerases. By means of extraction and subsequent gel filtration, several subspecies of complexes of 3'----5' exonuclease (E.C. 3.1.4.26) with DNA polymerases alpha, beta (E.C. 2.7.7.7) and some other proteins were isolated from chromatin, nucleoplasm, nuclear membrane, and cytosol. Complexes containing 3'----5' exonuclease manifest from 40 to 70% of total DNA polymerase activity revealed in different compartments of a hepatocyte. Molecular masses of the complexes amount from 250 to 1500 kDa They dissociate as a result of solution hydrophobization. DNA polymerase alpha activity enhances 5--8 folds during cell transition from G0 to S-period. The value of the ratio of 3'----5' exonuclease activity of different complexes to their DNA polymerase activity varies from 0.5 to 12. Other cases of discovery of the complexes of DNA polymerases with 3'----5' exonucleases are discussed. It is suggested that the absence of 3'----5' exonuclease active site in the DNA polymerase polypeptide is compensated by the complex formation of the corresponding enzymes.

Animals↗
PubMed1987

Nuclear endo-exonuclease of Neurospora crassa. Evidence for a role in DNA repair.

The major nuclease activity in nuclei of mycelia of Neurospora crassa has been identified as that of endoexonuclease, an enzyme purified and characterized previously from mitochondria and vacuoles which acts endonucleolytically on single-stranded DNA and RNA and possesses highly processive exonuclease activity with double-stranded DNA. Cross-contamination from the other organelles was eliminated as a source of the activity. Endo-exonuclease of nucleoplasm, chromatin, and nuclear matrix showed 80-100% cross-reaction with antisera raised to purified extranuclear endoexonuclease and was also strongly inhibited by 20 microM aurin tricarboxylic acid. In addition, it yielded some of the same-sized polypeptides on activity gel analysis. Nuclei also contained immunochemically cross-reactive trypsin-activable endo-exonuclease activity, a form of enzyme that was shown previously to occur in high amounts in the cytosol and in a tightly bound form associated with the mitochondrial inner membrane. Pretreatment of wild-type mycelia for 1 h with 4-16 micrograms/ml the DNA-damaging agent, 4-nitroquinoline-1-oxide (4-NQO), which caused about 50-80% growth inhibition, resulted in a dose-dependent loss of up to 80% of inactive endo-exonuclease from nuclei. At low doses of 4-NQO, this was accompanied by increases in the level of active enzyme. Nuclei of the DNA repair-deficient uvs-3 mutant were found to contain only 12% of the active enzyme and about 32% of inactive enzyme as that in wild-type nuclei. Mycelial growth of this mutant was 10 times more sensitive to 4-NQO than the wild-type. At a dose which resulted in equivalent growth inhibition, 4-NQO had no effect on the level of active endo-exonuclease in uvs-3 nuclei and caused an increase (over 30%) in the level of inactive enzyme. These data are consistent with a role of endo-exonuclease in the repair of nuclear DNA.

4-Nitroquinoline-1-oxide↗
PubMed1981

Herpes simplex virus type I DNA polymerase. Kinetic properties of the associated 3'-5' exonuclease activity and its role in araAMP incorporation.

An exonuclease activity copurified with herpes simplex virus type I (HSV-1) DNA polymerase through DNA-cellulose column chromatography and comigrated with DNA polymerase activity on nondenaturing gel electrophoresis at varied polyacrylamide concentrations. A gapped duplex DNA was the preferred substrate for this exonuclease activity since the hydrolytic activity on this type of DNA was much greater than the hydrolysis of either native or heat-denatured DNA. Using 3'-terminally labeled activated calf thymus DNA as substrate, the exonuclease activity was found to be activated by salt and spermidine in a manner identical with HSV-1 DNA polymerase. This activation was accompanied by increases in apparent Km and Vmax values of the activated DNA substrate. Phosphonoformic acid inhibited both DNA polymerase and exonuclease activities uncompetitively with respect to activated DNA and had a Ki of 2.4 microM at an ionic strength of 0.25 mu. Of the nucleoside 5'-monophosphates tested only the purine ribonucleotides inhibited the exonuclease activity. The inhibition was noncompetitive with respect to DNA, and GMP was about twice as potent as AMP or IMP. 9-beta-D-arabinosyladenine 5'-monophosphate (araAMP) could be incorporated into DNA by HSV-1 DNA polymerase; however, 9-beta-D-arabinosyladenine 5'-triphosphate would not replace dATP in supporting in vitro HSV-1 DNA synthesis. AraAMP incorporated into primer termini caused a significant decrease in the rate of subsequent primer elongation. These 3'-terminal araAMP residues could be removed by the HSV-1 DNA polymerase-associated exonuclease activity in a manner dependent on GMP concentration.

Arabinonucleotides↗
PubMed1980

Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma.

Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.

Animals↗
PubMed1994

3'-->5' exonuclease active site of phi 29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism.

The residues forming the 3'-->5' exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228; Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237). To understand their catalytic role, the residual exonuclease activity of mutants at these active site residues has been kinetically studied. The critical function of residues Asp12, Glu14, Asp66, and Asp169 is supported by a 10(5)-fold reduction in the exonuclease catalytic rate upon single mutation. Residue Tyr165 seems to play a secondary role in the exonuclease reaction based on the 10(2)-10(3)-fold reduced catalytic rate of mutants Y165F and Y165C. Most of the mutants were specially active in the presence of Mn2+ ions, which could be indicative of a direct involvement of these residues in a metal ion-assisted exonucleolytic reaction. The data obtained strongly suggest that the 3'-->5' exonuclease active site of phi 29 DNA polymerase is structurally and functionally similar to that of the Escherichia coli DNA polymerase I. In addition, these residues were also very important for the strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3'-->5' exonuclease.

Bacillus Phages↗
PubMedDate not supplied

[Contribution of 3'---5'-exonuclease from rat liver nuclei in precision of DNA synthesis, catalyzed by mammalian DNA polymerase alpha].

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of editing 3'-->5'exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5'exonucleases with molecular masses of 40 and 50 kDa have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from a poly [d(A-T)] template respectively 10- and 2-fold faster than the matched ones. Upon addition of any of these exonucleases to DNA polymerase alpha from rat liver or calf thymus, the fidelity of in vitro reproduction of primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, the levels of exonuclease and polymerase activities being approximately the same. The extrapolation of replication fidelity to cellular activities of the exonucleases and alpha-polymerase suggests that exonuclease proofreading augments the accuracy of DNA synthesis at least by three orders of magnitude.

Animals↗
PubMed1991

The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.

We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus. The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.

Amino Acid Sequence↗
PubMed1994

The 3'-->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae.

DNA polymerases II (epsilon) and III (delta) are the only nuclear DNA polymerases known to possess an intrinsic 3'-->5' exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase delta and epsilon 3'-->5' exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10(2) and 10(1), respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 x 10(3)-fold. Analysis of mutation rates of double mutants indicated that the 3'-->5' exonucleases of DNA polymerases delta and epsilon can act competitively and that, like the 3'-->5' exonuclease of DNA polymerase delta, the 3'-->5' exonuclease of DNA polymerase epsilon acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3'-->5' exonucleases of DNA polymerases delta and epsilon act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.

Amino Acid Sequence↗
PubMed1998

Age dependent decline in the 3'-->5' exonuclease activity involved in proofreading during DNA synthesis.

A 3'-->5' exonuclease found in rat liver excises mispaired nucleotides at the 3'-hydroxyl end of primer chains such as poly dA-d(T9-C). Consequently, the priming activity of the chain from which the mispaired base was cut is greatly increased during DNA synthesis. These results suggest that the 3'-->5' exonuclease acts as a proofreading enzyme during DNA synthesis. The activity of this 3'-->5' exonuclease in the liver of 24-month-old rats is approximately 30% lower than the activity found in 4-month-old rats. Furthermore, non-complementary nucleotide incorporations by DNA polymerases from aged rats are observed during DNA synthesis on poly dA-dT10. The number of misincorporations decreases in the presence of the 3'-->5' exonuclease, but not all errors are prevented even when DNA polymerase and 3'-->5' exonuclease are added at an activity ratio similar to that found in vivo. The data suggest that declines in both the fidelity of DNA polymerase and the 3'-->5' exonuclease activity related to proofreading during the aging process lead to a higher frequency of base misincorporations during DNA replication.

Aging↗
PubMed1999

Emergence of the active site of spleen exonuclease upon association of the two basic monomers of the tetrameric enzyme.

The 5'-->3' exonuclease from beef spleen is a 160-kDa tetramer consisting of four subunits of two types. Partial reduction of the tetramer led to one stable intermediate state of the enzyme with Mr = 80 kDa. The aim of this paper was to attribute the exonucleolytic activity to one of the two monomers, to the dimer or to the tetramer. The different forms of the exonuclease were separated by SDS-polyacrylamide gel electrophoresis, transferred on an Immobilon-P membrane and subsequently renaturated. Antibodies monospecific against each of the two monomers as well as against the dimer were isolated and their inhibitory effect on the holoenzyme determined. It was found that after renaturation the two monomers did not possess any exonuclease activity while the 80-kDa dimer showed a lower recovery of the specific activity of the enzyme (20.8+/-0.23 nkat/nmol, (n = 5)) in comparison with the 160-kDa tetramer (64.8+/-0.75 nkat/nmol (n = 5)). It was demonstrated that the antibodies monospecific against the dimer caused 53% maximum inhibition of the 160-kDa exonuclease. The antibodies monospecific against 25- and 55-kDa monomers did not inhibit the activity of the holoenzyme. No single-strand endonuclease activity of the spleen exonuclease was observed when using supercoiled Bluescript KS+ plasmid DNA as a substrate. This data suggest the emergence of an 80 kDa active form of beef spleen exonuclease upon association of two monomers of the tetrameric enzyme.

Animals↗
PubMed1991

Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction.

We present a kinetic partitioning mechanism by which the highly efficient 3'----5' exonuclease activity of T7 DNA polymerase maximizes its contribution to replication fidelity with minimal excision of correctly base-paired DNA. The elementary rate constants for the proposed mechanism have been measured directly from single-turnover experiments by using rapid chemical quench-flow techniques. The exonuclease activity of T7 DNA polymerase toward single-stranded DNA is quite fast (kx greater than 700 s-1). This rapid exonuclease is restrained with double-stranded DNA by a kinetic partitioning mechanism that favors the binding of the DNA to the polymerase site to prevent the rapid degradation of matched DNA and yet allows selective removal of mismatched DNAs. Both matched and mismatched DNAs bind tightly to the polymerase site, with approximately equal affinities, Kdp = 20 and 10 nM, respectively. Selective removal of the mismatch is governed by the rate of transfer of the DNA from the polymerase to the exonuclease site (kp----x). The rapid excision of matched DNA is limited by a slow transfer rate (kp----x = 0.2 s-1) from the polymerase to the exonuclease site relative to the rate of polymerization [kp = 300 s-1; Patel et al. (1991) Biochemistry (first of three papers in this issue)]. Removal of mismatched DNA is facilitated by its faster transfer rate (kp----x = 2.3 s-1) to the exonuclease site relative to the slow rate of polymerization over a mismatch [kpi = 0.012 s-1; Wong et al. (1991) Biochemistry (second of three papers in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2000

Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease.

Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.

Base Sequence↗