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PubMed1991

A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease.

The T5 D15 exonuclease purified from an overproducing strain of E. coli was shown to possess a low level of endonucleolytic activity specific for single-stranded DNA when assayed with 1-10 mM Mg2+ as co-factor. Endonuclease activity on double-stranded circular DNA could not be detected under these conditions. Nicked circular DNA was first gapped by the enzyme's exonucleolytic activity, creating a single-stranded region. This gapped substrate was then endonucleolytically cleaved and rapidly degraded. We show that a gapped and not a nicked substrate is required for this activity as previously suggested (Moyer, R. W. and Roth, C. T. 1977, J. Virol. 24, 177-193). The single-strand endonuclease activity could be selectively suppressed by using low concentrations of Mg2+ as co-factor (less than 1 mM), thus allowing nicked double-stranded circular DNA to be gapped to a single-stranded circular species. We also report on sequence similarities between the T5 exonuclease and several prokaryotic DNA polymerases.

Amino Acid Sequence↗

PubMed1998

Hex1: a new human Rad2 nuclease family member with homology to yeast exonuclease 1.

Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication. We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product. Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair. Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity. Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development. HEX1 gene equivalents were found in all vertebrates examined. The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors.

Amino Acid Sequence↗

PubMed2005

Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase beta and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity.

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase beta (pol beta), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol beta, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol beta, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol beta may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol beta. This enables pol beta to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.

Base Pair Mismatch↗

PubMed1999

Abasic site recognition mechanism by the Escherichia coli exonuclease III.

In order to elucidate the mechanism of AP site recognition by Echerichia coli exonuclease III (exoIII), site-directed mutagenesis of the Tyr109, the Trp212, and the Phe213 residues, which were conserved in the type II AP endonuclease from various organisms and located in the vicinity of the catalytic site, was performed. The exoIII-W212S mutant lacked any detectable AP endonuclease activity and binding ability to the duplex DNA containing an AP site, while the exoIII-Y109S and exoIII-F213W mutants retained a low level of activities (13% and 83%, respectively, compared with wild-type exoIII). This study suggests that the Trp212 is an important component for abasic site recognition by the E. coli exonuclease III.

Amino Acid Substitution↗

PubMed2003

Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.

Exonuclease 1 (Exo1) is a 5'-3' exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Analysis of Exo1(-/-) cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5' and 3' nick-directed repair. The repair defect in Exo1(-/-) cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1(-/-) animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition, Exo1(-/-) male and female mice were sterile because of a meiotic defect. Meiosis in Exo1(-/-) animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.

Animals↗

PubMed1993

High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.

Amino Acid Sequence↗

PubMed1994

The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.

The effect of phosphorothioate bonds on the hydrolytic activity of the 5'-->3' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5' biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophilic surface in the presence of salt.

Base Sequence↗

PubMed1994

Structure and function of the DNA repair enzyme exonuclease III from E. coli.

The three-dimensional structure of exonuclease III, the major AP DNA repair endonuclease of Escherichia coli, has been determined using x-ray crystallographic methods at 2.7 A resolution. The atomic model was fit to an electron density map calculated with phases obtained from three isomorphous heavy atom derivatives. The overall chain fold of exonuclease III is that of a compact alpha,beta-protein of dimensions 55 by 50 by 45 A. The pair of extended beta-pleated sheets pack against each other in an approximately parallel fashion to form the hydrophobic core of a four-layered sandwich structure. These beta sheets are flanked by four alpha-helices that form the outer two layers of the fold. The individual strands of the beta-sheets are in a mostly antiparallel configuration and are linked by extensive loop regions that connect adjoining strands. The structure contains internal symmetry with the two extended beta-sheets and four alpha-helices related by a pseudo-twofold axis running approximately down the center of the two sheets. This internal symmetry is not mirrored in the structure of the loop regions, nor is it detectable within the amino acid sequence. There is a "groove" between the beta-sheets at one end of the molecule that is bordered by several of the exposed loop regions and may be significant for DNA binding.

Amino Acid Sequence↗

PubMed2003

Sequence-dependent pausing of single lambda exonuclease molecules.

Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.

Bacteriophage lambda↗

PubMed1995

A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.

Exonuclease I (Exo I) from Schizosaccharomyces pombe, a 5'-->3' double-stranded DNA exonuclease, is induced during meiotic prophase I. The exo1 gene is a member of a family of related DNA repair genes, including RAD2/rad13/xpgc and YKL510/rad2, conserved from yeast to humans. An exo1 mutant displays a mutator phenotype and alters activity of the ade6-M387 marker effect. These results suggest that Exo I acts in a pathway that corrects mismatched base pairs.

Alleles↗

PubMed1984

Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli.

The stability of two ColE1-related plasmids (pRSF2124 and pMB9) was examined in strains of Escherichia coli multiply deficient in exonucleases I (sbcB), III (xthA), or V (recB recC). Any combination of exonuclease I, III, and V deficiency resulted in dramatically decreased stability of both pRSF2124 and pMB9. Inactivation of the RecF pathway by introducing either recF or recJ mutations to the recB recC subcB background resulted in nearly wild-type levels of stability for both plasmids. In contrast, the introduction of uvrD3 uvr-257, uvrE100, or recL152 into the recB21 recC22 sbcB15 strain did not affect plasmid stability. Furthermore, the amount of plasmid DNA recovered from pRSF2124 or pMB9 transformants of a xthA1 sbcB15 strain was strikingly reduced relative to that of a wild-type control. Taken together, these results suggest that some aspect of DNA repair is required for stable maintenance of ColE1-related plasmids in E. coli.

Bacteriocin Plasmids↗

PubMed1994

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

Bacterial Proteins↗

PubMed1998

Mismatch repair in Escherichia coli cells lacking single-strand exonucleases ExoI, ExoVII, and RecJ.

In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.

Bacterial Proteins↗

PubMed2006

Single-strand-specific exonucleases prevent frameshift mutagenesis by suppressing SOS induction and the action of DinB/DNA polymerase IV in growing cells.

Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI- ExoVII- cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI- ExoVII- cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.

DNA Polymerase beta↗

PubMed1983

High-resolution characterization of herpes simplex virus type 1 transcripts encoding alkaline exonuclease and a 50,000-dalton protein tentatively identified as a capsid protein.

Four partially overlapping mRNAs (1.9, 2.3, 3.9, and 4.5 kilobases [kb]) were located between 0.16 and 0.19 map units on the herpes simplex virus type 1 genome. Their direction of transcription was found to be from right to left. The 2.3-kb mRNA was found to be early (beta), whereas the others were late (beta gamma). Partial sequence analysis of the DNA encoding these genes indicated that the promoter for the 2.3-kb mRNA shares structural features with other early (beta) promoters. In vitro translation of hybrid-selected mRNA indicated that among the proteins these mRNAs encode are an 82,000-dalton (d) polypeptide reactive with a monoclonal antibody against herpes simplex virus type 2 alkaline exonuclease and a 50,000-d polypeptide weakly reactive with a polyclonal antibody made against the capsid protein VP19C. Further experiments suggested that the 2.3-kb mRNA encodes the 82,000-d polypeptide, whereas one (or both) of the larger mRNAs encodes the 50,000-d protein. A novel finding was that the 1.9-kb mRNA appears to share part of the translational reading frame for alkaline exonuclease, but any polypeptide it encodes does not react with the monoclonal antibody to this enzyme.

Base Sequence↗

PubMed1984

Physical mapping of the herpes simplex virus type 2 nuc- lesion affecting alkaline exonuclease activity by using herpes simplex virus type 1 deletion clones.

The nuc- lesion affecting alkaline exonuclease activity in the herpes simplex virus type 2 (HSV-2) mutant ts1348 had previously been mapped to the EcoRI-D restriction enzyme fragment of HSV-1. Eight clones with deletions representing most of HSV-1 EcoRI fragment D were selected with lambda gtWES hybrids. These clones were tested for their ability to rescue the alkaline exonuclease activity of HSV-2 nuc- ts1348 virus. The sequences colinear with the HSV-2 nuc- lesion were found to map between 0.169 and 0.174 map units on the HSV-1 Patton genome, representing an 0.8-kilobase-pair region that is 12.9 to 13.7 kilobase pairs from the left end of HSV-1 EcoRI fragment D.

Animals↗

PubMed1989

Locations of amino acid substitutions in bacteriophage T4 tsL56 DNA polymerase predict an N-terminal exonuclease domain.

The amino acid substitutions responsible for the temperature-sensitive (ts) and mutator phenotypes of the classical bacteriophage T4 DNA polymerase mutant tsL56 were determined. tsL56 DNA polymerase has two mutations in the 5' end of the DNA polymerase gene (g43) that produce two amino acid substitutions: codon 89, alanine to threonine, and codon 363, aspartate to asparagine. Both mutations are required for the strong ts and mutator phenotypes. The increased error rate of the tsL56 DNA polymerase is due to a reduction in 3'----5' exonuclease activity relative to polymerase activity (N. Muzyczka, R. L. Poland, and M. J. Bessman, J. Biol. Chem. 247:7116-7122, 1972). Thus, the locations of the tsL56 mutations suggest that the 3'----5' exonuclease domain resides in the N-terminal region. Several other ts DNA polymerase mutant strains isolated with tsL56 also have mutator or antimutator phenotypes. The nucleotide changes in these important mutant strains were also determined. This mutant collection, combined with collections of g43 amber mutants and mutants selected on the basis of a strong mutator phenotype (L. J. Reha-Krantz, J. Mol. Biol. 202:711-724, 1988), contains nearly 70 different DNA polymerase mutations. The numerous T4 DNA polymerase mutations are valuable for DNA polymerase structure-function and fidelity studies.

Base Sequence↗

PubMed1997

The poly(A) tail inhibits the assembly of a 3'-to-5' exonuclease in an in vitro RNA stability system.

We have developed an in vitro system which faithfully reproduces several aspects of general mRNA stability. Poly(A)- RNAs were rapidly and efficiently degraded in this system with no detectable intermediates by a highly processive 3'-to-5' exonuclease activity. The addition of a poly(A) tail of at least 30 bases, or a 3' histone stem-loop element, specifically stabilized these transcripts. Stabilization by poly(A) required the interaction of proteins with the poly(A) tail but did not apparently require a 3' OH or interaction with the 5' cap structure. Finally, movement of the poly(A) tract internal to the 3' end caused a loss of its ability to stabilize transcripts incubated in the system but did not affect its ability to interact with poly(A) binding proteins. The requirement for the poly(A) tail to be proximal to the 3' end indicates that it mediates RNA stability by blocking the assembly, but not the action, of an exonuclease involved in RNA degradation in vitro.

Exoribonucleases↗