DNA Exonuclease Activity Research Articles

An exonucleolytic activity of human apurinic/apyrimidinic endonuclease on 3' mispaired DNA.

Human apurinic/apyrimidinic endonuclease (APE1) is an essential enzyme in DNA base excision repair that cuts the DNA backbone immediately adjacent to the 5' side of abasic sites to facilitate repair synthesis by DNA polymerase beta (ref. 1). Mice lacking the murine homologue of APE1 die at an early embryonic stage. Here we report that APE1 has a DNA exonuclease activity on mismatched deoxyribonucleotides at the 3' termini of nicked or gapped DNA molecules. The efficiency of this activity is inversely proportional to the gap size in DNA. In a base excision repair system reconstituted in vitro, the rejoining of nicked mismatched DNA depended on the presence of APE1, indicating that APE1 may increase the fidelity of base excision repair and may represent a new 3' mispaired DNA repair mechanism. The exonuclease activity of APE1 can remove the anti-HIV nucleoside analogues 3'-azido-3'-deoxythymidine and 2',3'-didehydro-2', 3'-dideoxythymidine from DNA, suggesting that APE1 might have an impact on the therapeutic index of antiviral compounds in this category.

Structure and Mechanism of E. coli Rnase.

INTRODUCTION. RNase T is one of eight distinct 3’ to 5’ exoribonucleases identified in Escherichia coli[1]. It belongs to the DEDD exonuclease superfamily[2] characterized by common motifs consisting of four invariant acidic residues, which in DNA polymerases were shown to form the exonuclease active site[3]. RNase T plays an important role in stable RNA metabolism, including tRNA end turnover and 3’ maturation of many stable RNAs[1]. RNase T proteins are small polypeptides of about 220 amino acids. They are closely related to the proofreading domains/subunits of bacterial DNA polymerase III, and interestingly, E. coli RNase T also displays strong DNA exonuclease activity[4,5]. E. coli RNase T must form a homodimer in order to function[1].

DNase I footprinting and enhanced exonuclease function of the bipartite Werner syndrome protein (WRN) bound to partially melted duplex DNA.

Werner syndrome is a premature aging and cancer-prone hereditary disorder caused by deficiency of the WRN protein that harbors 3' -->5' exonuclease and RecQ-type 3' --> 5' helicase activities. To assess the possibility that WRN acts on partially melted DNA intermediates, we constructed a substrate containing a 21-nucleotide noncomplementary region asymmetrically positioned within a duplex DNA fragment. Purified WRN shows an extremely efficient exonuclease activity directed at both blunt ends of this substrate, whereas no activity is observed on a fully duplex substrate. High affinity binding of full-length WRN protects an area surrounding the melted region of the substrate from DNase I digestion. ATP binding stimulates but is not required for WRN binding to this region. Thus, binding of WRN to the melted region underlies the efficient exonuclease activity directed at the nearby ends. In contrast, a WRN deletion mutant containing only the functional exonuclease domain does not detectably bind or degrade this substrate. These experiments indicate a bipartite structure and function for WRN, and we propose a model by which its DNA binding, helicase, and exonuclease activities function coordinately in DNA metabolism. These studies also suggest that partially unwound or noncomplementary regions of DNA could be physiological targets for WRN.

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.

Nuclease activity is essential for RecBCD recombination in Escherichia coli.

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).

Interactions between the Werner Syndrome Helicase and DNA Polymerase δ Specifically Facilitate Copying of Tetraplex and Hairpin Structures of the d(CGG)n Trinucleotide Repeat Sequence

Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

9] DNA polymerases from hyperthermophiles

Publisher Summary With the emergence of the polymerase chain reaction (PCR), considerable attention has been focused on isolating and characterizing the DNA polymerases of thermophiles. Numerous laboratory techniques, including PCR, RT-PCR (reverse transcription-PCR), isothermal amplification, DNA sequencing, mutagenesis, and cloning, rely on thermostable DNA polymerases for carrying out DNA synthesis in vitro at high temperatures. DNA polymerases perform essential roles in the replication and repair of genetic material. In addition to thermostable eubacterial enzymes, a number of DNA polymerases from hyperthermophilic archaea have been isolated and characterized. The most abundant DNA polymerase(s) in archaea are homologs of Family B DNA polymerases. This chapter presents a compilation of methods used to assay various activities associated with thermostable DNA polymerases, including DNA polymerase activity (nucleotide incorporation), processivity, strand displacement, and exonuclease activity. The chapter describes modifications to the nucleotide incorporation assay that allow measurements of thermostability, steady-state kinetic parameters, nucleotide analog incorporation, and reverse transcriptase activity. In addition, the chapter provides a brief discussion regarding polymerase error rate determinations.

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.

Facilitated Loading of RecA Protein Is Essential to Recombination by RecBCD Enzyme

Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme. However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.

Plant homologue of flap endonuclease-1: molecular cloning, characterization, and evidence of expression in meristematic tissues.

Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. We isolated a 1.4 kb cDNA from rice (Oryza sativa), termed OsFEN-1, encoding a protein which shows homology with the eukaryotic FEN-1 proteins. OsFEN-1 protein was overexpressed in Escherichia coli and purified to near homogeneity. DNA cleavage analysis using different branched DNA structures indicated that OsFEN-1 protein possesses both 5'-flap endonuclease and 5' to 3' double-stranded DNA exonuclease activities. OsFEN-1 protein incises a 5'-flap and 5'-pseudo Y structure one base 3' of the branched point in the duplex region. The enzymatic properties indicated that we succeeded in obtaining the gene and the protein of a plant counterpart of FEN-1. OsFEN-1 transcripts were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively. No expression was detected in mature leaves although the leaves were exposed to UV. We analyzed the spatial distribution pattern of OsFEN-1 transcripts by in situ hybridization. In the shoot apex, OsFEN-1 mRNA was abundant in the shoot apical meristem, tiller bud, leaf primordia, ligule primordia and marginal meristem of young leaves. In the roots, the transcript accumulated to high levels in the root apical meristem. Our results indicate that OsFEN-1 is expressed in tissues rich in proliferating cells, and its expression may be required for cell growth and organ formation.

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.

5' to 3' Single Strand DNA Exonuclease Activity in a Preparation of Human Ku Protein

5' to 3' Single Strand DNA Exonuclease Activity in a Preparation of Human Ku Protein

Physical and Functional Interaction between p53 and the Werner's Syndrome Protein

Werner's syndrome is a human autosomal recessive disorder leading to premature aging. The mutations responsible for this disorder have recently been localized to a gene (WRN) encoding a protein that possesses DNA helicase and exonuclease activities. Patients carrying WRN gene mutations exhibit an elevated rate of cancer, accompanied by increased genomic instability. The latter features are also characteristic of the loss of function of p53, a tumor suppressor that is very frequently inactivated in human cancer. Moreover, changes in the activity of p53 have been implicated in the onset of cellular replicative senescence. We report here that the WRN protein can form a specific physical interaction with p53. This interaction involves the carboxyl-terminal part of WRN and the extreme carboxyl terminus of p53, a region that plays an important role in regulating the functional state of p53. A small fraction of WRN can be found in complex with endogenous p53 in nontransfected cells. Overexpression of WRN leads to augmented p53-dependent transcriptional activity and induction of p21(Waf1) protein expression. These findings support the existence of a cross-talk between WRN and p53, which may be important for maintaining genomic integrity and for preventing the accumulation of aberrations that can give rise to premature senescence and cancer.

Active-site mutations in the Xrn1p exoribonuclease of Saccharomyces cerevisiae reveal a specific role in meiosis.

Xrn1p of Saccharomyces cerevisiae is a major cytoplasmic RNA turnover exonuclease which is evolutionarily conserved from yeasts to mammals. Deletion of the XRN1 gene causes pleiotropic phenotypes, which have been interpreted as indirect consequences of the RNA turnover defect. By sequence comparisons, we have identified three loosely defined, common 5'-3' exonuclease motifs. The significance of motif II has been confirmed by mutant analysis with Xrn1p. The amino acid changes D206A and D208A abolish singly or in combination the exonuclease activity in vivo. These mutations show separation of function. They cause identical phenotypes to that of xrn1Delta in vegetative cells but do not exhibit the severe meiotic arrest and the spore lethality phenotype typical for the deletion. In addition, xrn1-D208A does not cause the severe reduction in meiotic popout recombination in a double mutant with dmc1 as does xrn1Delta. Biochemical analysis of the DNA binding, exonuclease, and homologous pairing activity of purified mutant enzyme demonstrated the specific loss of exonuclease activity. However, the mutant enzyme is competent to promote in vitro assembly of tubulin into microtubules. These results define a separable and specific function of Xrn1p in meiosis which appears unrelated to its RNA turnover function in vegetative cells.

Human Exonuclease 1 Functionally Complements Its Yeast Homologues in DNA Recombination, RNA Primer Removal, and Mutation Avoidance

Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Delta mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.

Different regulation of the p53 core domain activities 3'-to-5' exonuclease and sequence-specific DNA binding.

In this study we further characterized the 3'-5' exonuclease activity intrinsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-terminal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activity by at least 10-fold, indicating that this activity, like sequence-specific DNA binding, is negatively regulated by the C-terminal basic regulatory domain of p53. However, treatments which activated sequence-specific DNA binding of p53, like binding of the monoclonal antibody PAb421, which recognizes a C-terminal epitope on p53, or a higher phosphorylation status, strongly inhibited the p53 exonuclease activity. This suggests that at least on full-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Following up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombination intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such substrates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to the binding assay indeed started the p53 exonuclease and promoted rapid degradation of the bound, but not of the unbound, substrate, indicating that specifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.

Rejoining of DNA double-strand breaks in vitro by single-strand annealing.

Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in vertebrate cells. Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku. While the former pathway has been characterised in some detail, only little is known about the latter error-prone. We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs. End-joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single-strand annealing (SSA). Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase epsilon, FEN-1 endonuclease, and exonuclease activities of 5'-3' and 3'-5' directionality. We show that these activities are able to correctly process proposed intermediates of SSA. Interestingly, neither Ku nor the associated DNA-dependent protein kinase were detected, indicating that the mechanism can dispense with Ku. Our findings provide evidence for the existence of an error-prone NHEJ pathway that creates deletions by microhomology-driven SSA.

Werner Syndrome Protein: I. DNA HELICASE AND DNA EXONUCLEASE RESIDE ON THE SAME POLYPEPTIDE

Werner Syndrome (WS) is a human progeroid disorder characterized by genomic instability. The gene defective in WS encodes a 3' --> 5' DNA helicase (Gray, M. D., Shen, J.-C., Kamath-Loeb, A. S., Blank, A. , Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A.(1997) Nat. Genet. 17, 100-103). Sequence alignment analysis identified an N-terminal motif in WRN that is homologous to several exonucleases. Using combined molecular genetic, biochemical, and immunochemical approaches, we demonstrate that WRN also exhibits an integral DNA exonuclease activity. First, whereas wild-type recombinant WRN possesses both helicase and exonuclease activities, mutant WRN lacking the nuclease domain does not display exonucleolytic activity. In contrast, WRN proteins with defective helicase activity are active in exonucleolytic digestion of DNA. Second, the exonuclease co-purifies with the 160-kDa WRN protein and its associated DNA helicase and ATPase activities through successive steps of ion exchange and affinity chromatography, suggesting that all three activities are physically associated. Lastly, anti-WRN antiserum specifically co-precipitates the WRN helicase and exonuclease activities indicating that both activities reside on the same antigenic WRN polypeptide. The association of an exonuclease with WRN distinguishes it from other RecQ homologs and raises the possibility that the distinct phenotypic characteristics of WS may be due in part to a defective exonuclease.

Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: requirement for DNA end processing.

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T4 2(-) of about 200-fold (phage T4 2(-) has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb+) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.