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PubMed2002

The 3' 5' exonucleases.

Over the past few years, several new 3' 5' exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3' 5' exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3' 5' exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.

Amino Acid Sequence↗
PubMed1994

2-Chloro-2'-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3'-->5' exonucleases.

2-Chloro-2'-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3'-->5' exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates. Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3'-->5' exonuclease, but only in the vicinity of the ClAde sites. Approx. 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA. Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3'-->5' exonucleases, incorporated ClAde enhances strand degradation of duplex DNA.

Adenine↗
PubMed1997

Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III.

Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-alpha,beta unsaturated aldehyde that prevents DNA-repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the alpha,beta unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.

Apurinic Acid↗
PubMed1998

Purification and properties of the 5'-3' exonuclease D190-->a mutant of DNA polymerase I from Streptococcus pneumoniae.

A D190-->A mutation was introduced at the 5'-3' exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site-directed mutagenesis of the polA gene. Comparison of the S. pneumoniae DNA polymerase I, its polymerase domain, and the [Ala190]exonuclease mutant revealed that the mutant polypeptide retains the polymerase activity of the parental enzyme and displayed the strand-displacement activity of its polymerase domain. However, introduction of the mutation resulted in a 2500-fold reduction of the 5'-3' exonuclease catalytic rate compared with the wild-type enzyme. Moreover, the mutation at the Asp190 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5'-3' exonucleolytic activity. These results provide experimental support for a direct involvement of this aspartic acid residue in a metal-assisted 5'-3' exonucleolytic reaction in type-I-like bacterial DNA polymerases and related bacteriophage 5'-3' exonucleases.

Binding Sites↗
PubMed2006

RecA-independent recombination is efficient but limited by exonucleases.

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecA-independent recombination in Escherichia coli is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecA-dependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI(-) RecJ(-) strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that E. coli encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors.

Crossing Over, Genetic↗
PubMed1971

Genetic recombination in Escherichia coli: the role of exonuclease I.

The indirect suppression of recB(-) and recB(-)recC(-) mutations by the sbcB(-) allele is caused by the loss of a nuclease active on denatured DNA. Results from enzyme purifications and studies with a specific antiserum demonstrate that the activity present in sbcB(+) strains, and lost in sbcB(-) strains, is exonuclease I. It is likely that sbcB is the structural gene for exonuclease I. The loss of exonuclease I activity restores the recombination proficiency of Escherichia coli cells that has been lost by mutations in the recB and/or recC genes. This indicates that in the absence of the recB-recC-determined enzyme, exonuclease I prevents recombination. Hypothetical pathways illustrating this conclusion are presented.

Alleles↗
PubMed1984

A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme.

DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases. The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene. Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit. To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme. We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits. We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E. coli and DNA polymerase of phage T4. This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.

DNA Polymerase III↗
PubMed1985

Exonuclease III recognizes urea residues in oxidized DNA.

Escherichia coli exonuclease III was found to be associated with an activity that recognizes urea residues in DNA but not thymine glycol residues from which the urea residues were prepared. This activity was not due to a contaminating activity such as endonuclease III since urea-containing DNA was a competitive inhibitor of exonuclease III when apurinic DNA was used as a substrate and vice versa. The apparent kinetic constants for both the substrate and inhibitor were determined. Like its apurinic activity, exonuclease III activity against urea residues was endonucleolytic, nicking on the 5' side of the damage and having an optimal Mg2+ concentration between 2 and 10 mM. Also, the enzyme recognized alkali-stable damages produced in DNA by H2O2 in vitro. We suggest that it may be this activity of exonuclease III that accounts for its biological role in vivo.

DNA↗
PubMed1989

Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos.

The mitochondrial DNA polymerase from Drosophila embryos lacks dNTP turnover activity. However, a potent 3'----5' exonuclease activity can be detected by a specific assay in which the exonuclease excises mispaired nucleotides at the 3' termini of primed synthetic and natural DNA templates. The excision of a mispaired nucleotide occurs at a significantly greater rate than excision of a correctly paired nucleotide and, under conditions of DNA synthesis, hydrolysis of a mispaired terminal nucleotide occurs prior to primer extension. The 3'----5' exonuclease copurifies quantitatively with DNA polymerase gamma and cosediments with the nearly homogeneous enzyme under native conditions. These results suggest that the 3'----5' exonuclease provides a proofreading function to enhance the fidelity of DNA synthesis during Drosophila mitochondrial DNA replication.

Animals↗
PubMed1999

Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.

Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.

Amino Acid Sequence↗
PubMed1997

The 5'-exonuclease activity of bacteriophage T4 RNase H is stimulated by the T4 gene 32 single-stranded DNA-binding protein, but its flap endonuclease is inhibited.

Bacteriophage T4 RNase H is a 5'- to 3'-nuclease that has exonuclease activity on RNA.DNA and DNA.DNA duplexes and can remove the pentamer RNA primers made by the T4 primase-helicase (Hollingsworth, H. C., and Nossal, N. G. (1991) J. Biol. Chem. 266, 1888-1897; Hobbs, L. J., and Nossal, N. G. (1996) J. Bacteriol. 178, 6772-6777). Here we show that this exonuclease degrades duplex DNA nonprocessively, releasing a single oligonucleotide (nucleotides 1-4) with each interaction with the substrate. Degradation continues nonprocessively until the enzyme stops 8-11 nucleotides from the 3'-end of the substrate. T4 gene 32 single-stranded DNA-binding protein strongly stimulates the exonuclease activity of T4 RNase H, converting it into a processive nuclease that removes multiple short oligonucleotides with a combined length of 10-50 nucleotides each time it binds to the duplex substrate. 32 protein must bind on single-stranded DNA behind T4 RNase H for processive degradation. T4 RNase H also has a flap endonuclease activity that cuts preferentially on either side of the junction between single- and double-stranded DNA in flap and fork DNA structures. In contrast to the exonuclease, the endonuclease is inhibited completely by 32 protein binding to the single strand of the flap substrate. These results suggest an important role for T4 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle.

Bacteriophage T4↗
PubMed2001

p53 Modulates the exonuclease activity of Werner syndrome protein.

Werner syndrome (WS) is characterized by the early onset of symptoms of premature aging, cancer, and genomic instability. The molecular basis of the defects is not understood but presumably relates to the DNA helicase and exonuclease activities of the protein encoded by the WRN gene that is mutated in the disease. The attenuation of p53-mediated apoptosis in WS cells and reported physical interaction between WRN and the tumor suppressor p53 suggest that p53 and WRN functionally interact in a pathway necessary for the normal cellular response. In this study, we have demonstrated that p53 inhibits the exonuclease activity of the purified full-length recombinant WRN protein. p53 did not have an effect on a truncated amino-terminal WRN fragment that retains exonuclease activity but lacks the physical interaction domain for p53 located in the carboxyl terminus. Two naturally occurring p53 mutants found in human cancer displayed a reduced ability to inhibit WRN exonuclease activity. In cells arrested in S phase with hydroxyurea, WRN exits the nucleolus and colocalizes with p53 in the nucleoplasm. The regulation of WRN function by p53 is likely to play an important role in the maintenance of genomic integrity and prevention of cancer and other clinical symptoms associated with WS.

Adenosine Triphosphatases↗
PubMed2006

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family.

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.

Bacteriophage T4↗
PubMed1999

5' to 3' single strand DNA exonuclease activity in a preparation of human Ku protein.

We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line. The enzyme removes 5' single-strand extensions from duplex DNA molecules. The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase. The exonuclease activity does not require divalent cations. The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed.

Animals↗
PubMed2002

CCR4, a 3'-5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase.

The CCR4-NOT complex from Saccharomyces cerevisiae is a general transcriptional regulatory complex. The proteins of this complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation. The evolutionarily conserved CCR4 protein, which is part of the cytoplasmic deadenylase, contains a C-terminal domain that displays homology to an Mg2+-dependent DNase/phosphatase family of proteins. We have analyzed the putative enzymatic properties of CCR4 and have found that it contains both RNA and single-stranded DNA 3'-5' exonuclease activities. CCR4 displays a preference for RNA and for 3' poly(A) substrates, implicating it as the catalytic component of the cytoplasmic deadenylase. Mutations in the key, conserved catalytic residues in the CCR4 exonuclease domain abolished both its in vitro activities and its in vivo functions. Importantly, CCR4 was active as a monomer and remained active in the absence of CAF1, which links CCR4 to the remainder of the CCR4-NOT complex components. These results establish that CCR4 and most probably other members of a widely distributed CCR4-like family of proteins constitute a novel class of RNA-DNA exonucleases. The various regulatory effects of the CCR4-NOT complex on gene expression may be executed in part through these CCR4 exonuclease activities.

Amino Acid Sequence↗
PubMed1997

Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease.

Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.

Base Sequence↗
PubMed1999

Werner syndrome helicase contains a 5'-->3' exonuclease activity that digests DNA and RNA strands in DNA/DNA and RNA/DNA duplexes dependent on unwinding.

We show that WRN helicase contains a unique 5'-->3' exonuclease activity in the N-terminal region. Adeletion mutant lacking 231 N-terminal amino acid residues, made in a baculovirus system, did nothave this activity, while it showed ATPase and DNA helicase activities. This exonuclease activity was co-precipitated with the helicase activity using monoclonal antibodies specific to WRN helicase, indicating that it is an integral component with WRN helicase. The exonuclease in WRN helicase does not digest free single-stranded DNA or RNA, but it digests a strand in the duplex DNA or an RNA strand in a RNA/DNA heteroduplex in a 5'-->3' direction dependent on duplex unwinding. The digestion products were identified as 5'-mononucleotides. Our data show that WRN helicase needs a single-stranded 3' overhang region for efficient binding and unwinding of duplex molecules, while blunt-ended or 5' overhang duplex molecules were hardly unwound. These findings suggest that the WRN helicase and integral 5'-->3' exonuclease activities are involved in preventing a hyper-recombination by resolving entangled structures of DNA and RNA/DNA heteroduplexes that may be generated during rep-lication, repair and/or transcription.

Animals↗
PubMed2000

Characterization of the human and mouse WRN 3'-->5' exonuclease.

Werner's syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3'-->5' DNA helicase activity. We recently showed that hWRN is also a 3'-->5' exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3' recessed strands of double-stranded DNA or a DNA-RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3' protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3' recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3'-->5' exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.

Amino Acid Sequence↗