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PubMed1985

DNA filter retention assay for exonuclease activities. Application to the analysis of processivity of phage T5 induced 5'-exonuclease.

The 5'-exonuclease of phage T5 has been purified nearly to homogeneity by using a simple and fast procedure. The kinetic properties of the purified enzyme have been studied by using a new sensitive assay based upon retention by nitrocellulose filters of DNA with short protruding single-stranded ends. The enzyme is specifically stimulated by KCl. Its Km is 2.2 X 10(-7) M at 30 degrees C, and its turnover number is 0.33 DNA molecule transformed per minute. The filter retention assay shows that the T5 exonuclease acts by a semiprocessive mechanism, removing from DNA ends about 30 nucleotides on the average per cycle. The degree of enzyme processivity increases with increasing magnesium concentrations.

DNA, Viral↗
PubMed2001

Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo-exonuclease activity in T5 exonuclease.

T5 5'-3' exonuclease is a member of a homologous group of 5' nucleases which require divalent metal co-factors. Structural and biochemical studies suggest that single-stranded DNA substrates thread through a helical arch or hole in the protein, thus bringing the phosphodiester backbone into close proximity with the active site metal co-factors. In addition to the expected use of Mg(2+), Mn(2+) and Co(2+) as co-factors, we found that divalent zinc, iron, nickel and copper ions also supported catalysis. Such a range of co-factor utilisation is unusual in a single enzyme. Some co-factors such as Mn(2+) stimulated the cleavage of double-stranded closed-circular plasmid DNA. Such endonucleolytic cleavage of circular double-stranded DNA cannot be readily explained by the threading model proposed for the cleavage of substrates with free 5'-ends as the hole observed in the crystal structure of T5 exonuclease is too small to permit the passage of double-stranded DNA. We suggest that such a substrate may gain access to the active site of the enzyme by a process which does not involve threading.

Base Sequence↗
PubMed2002

The crystal structure of exonuclease RecJ bound to Mn2+ ion suggests how its characteristic motifs are involved in exonuclease activity.

RecJ, a 5' to 3' exonuclease specific for single-stranded DNA, functions in DNA repair and recombination systems. We determined the crystal structure of RecJ bound to Mn(2+) ion essential for its activity. RecJ has a novel fold in which two domains are interconnected by a long helix, forming a central groove. Mn(2+) is located on the wall of the groove and is coordinated by conserved residues characteristic of a family of phosphoesterases that includes RecJ proteins. The groove is composed of residues conserved among RecJ proteins and is positively charged. These findings and the narrow width of the groove indicate that the groove binds single- instead of double-stranded DNA.

Amino Acid Motifs↗
PubMed1999

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

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

Base Sequence↗
PubMed1996

Functional consequences and exonuclease kinetic parameters of point mutations in bacteriophage T4 DNA polymerase.

Three groups of T4 DNA polymerase mutants were prepared and characterized. In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I. Two divalent metal ions required for catalyzing the 3'-5' exonuclease reaction are ligated by carboxyl groups from these conserved Asp and Glu residues. The Ala and Asn replacements have a profound effect on the exonuclease activity of T4 DNA polymerase and also have a significant, but less pronounced influence on its polymerase activity which is located in a domain distal to the exonuclease region. The kcat values for the exonuclease reaction were reduced by 3-4 orders of magnitude by these replacements, but the values of Km(app) did not differ greatly from the wild-type enzyme. The second group consists of replacements of other residues, that are conserved in the exonuclease domain of eukaryotic DNA polymerases, but do not contribute to divalent metal ion coordination. Many of these alterations resulted in decreased exonuclease and/or polymerase activity. Mutants in the third group have substitutions of conserved residues in the polymerase domain which diminished polymerase and altered exonuclease activities. Our results, combined with structural data on crystals of protein N388, a truncated form of T4 DNA polymerase (Wang et al., 1996), show that: (i) the reduction in the relative specific exonuclease activities of mutants in the first group was significantly less than that of mutants in the Klenow fragment, despite the nearly identical geometric arrangement of the metal liganding groups in two proteins; (ii) altered residues, that affect exonuclease and/or polymerase activities in mutants of the second group, cluster within a small area of the exonuclease domain, suggesting that this area may be directly or indirectly involved in polymerase activity; (iii) mutations in the third group, which affect polymerase and exonuclease activities, may participate in DNA and dNTP binding. Our results point to the functional interdependence of the polymerase and exonuclease domains in T4 DNA polymerase, a property not observed with the Klenow fragment.

Amino Acid Sequence↗
PubMed2003

p53-associated 3'-->5' exonuclease activity in nuclear and cytoplasmic compartments of cells.

The tumor suppressor protein p53 plays an important role in maintenance of the genomic integrity of cells. p53 possesses an intrinsic 3'-->5' exonuclease activity. p53 was found in the nucleus and in the cytoplasm of the cell. In order to evaluate the subcellular location and extent of p53-associated 3'--> 5' exonuclease activity, we established an in vitro experimental system of cell lines with different nuclear/cytoplasmic distribution of p53. Nuclear and cytoplasmic extracts obtained from LCC2 cells (expressing a high level of cytoplasmic wild-type p53), MCF-7 cells (expressing a high level of wild-type nuclear p53), MDA cells (expressing mutant p53) and H1299 cells (p53-null) were subjected to the analysis of exonuclease activity. Interestingly, 3'-->5' exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all cell lines tested, exerted a low level of exonuclease activity. Cytoplasmic extracts of LCC2 cells, with a high level of wild-type p53, showed an enhanced exonuclease activity in comparison to those expressing either a low level of wild-type p53 (in MCF-7 cells) or the mutant p53 (in MDA cells). Evidence that exonuclease function detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: First, this activity closely parallels with levels and status of endogenous cytoplasmic p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the exonuclease activity. Third, the observed 3'-->5' exonuclease in cytoplasmic fraction of LCC2 cells displays identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include: (a) substrate specificity; exonuclease hydrolyzes single-stranded DNA in preference to double-stranded DNA and RNA/DNA template-primers, (b) efficient excision of 3'-terminal mispairs from DNA/DNA and RNA/DNA substrates, (c) the preferential excision of purine-purine mispairs over purine-pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by excision of mismatched nucleotides from the nascent DNA strand. Taken together, the data demonstrate that wild-type p53 in cytoplasm, in its noninduced state, is functional; it displays intrinsic 3'-->5' exonuclease activity. The possible role of p53-associated 3'-->5' exonuclease activity in DNA repair in nucleus and cytoplasm is discussed.

Breast Neoplasms↗
PubMed1991

Identification and characterization of a 3' to 5' exonuclease associated with spinach chloroplast DNA polymerase.

Spinach chloroplast DNA polymerase was shown to copurify with a 3' to 5' exonuclease activity during DEAE-cellulose, hydroxylapatite, and heparin-agarose column chromatography. In addition, both activities comigrated during nondenaturing polyacrylamide gel electrophoresis and cosedimented through a glycerol gradient with an apparent molecular weight of 105,000. However, two forms of exonuclease activity were detected following velocity sedimentation analysis. Form I constituted approximately 35% of the exonuclease activity and was associated with the DNA polymerase, whereas the remaining activity (form II) was free of DNA polymerase and exhibited a molecular weight of approximately 26,500. Resedimentation of form I exonuclease generated both DNA polymerase associated and DNA polymerase unassociated forms of the exonuclease, suggesting that polymerase/exonuclease dissociation occurred. The exonuclease activity (form I) was somewhat resistant to inhibition by N-ethylmaleimide, whereas the DNA polymerase activity was extremely sensitive. Using in situ detection following SDS-polyacrylamide activity gel electrophoresis, both form I and II exonucleases were shown to reside in a similar, if not identical, polypeptide of approximately 20,000 molecular weight. Both form I and II exonucleases were equally inhibited by NaCl and required 7.5 mM MgCl2 for optimal activity. The 3' to 5' exonuclease excised deoxyribonucleoside 5'-monophosphates from both 3'-terminally matched and 3'-terminally mismatched primer termini. In general, the exonuclease preferred to hydrolyze mismatched 3'-terminal nucleotides as determined from the Vmax/Km ratios for all 16 possible combinations of matched and mismatched terminal base pairs. These results suggest that the 3' to 5' exonuclease may be involved in proofreading errors made by chloroplast DNA polymerase.

Ammonium Sulfate↗
PubMed2003

The enzymatic basis of processivity in lambda exonuclease.

Lambda exonuclease is a highly processive 5'-->3' exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by lambda exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of lambda biology, lambda exonuclease enzymology and the availability of the X-ray crystallographic structure of lambda exonuclease make it a suitable model to dissect the mechanisms of processivity. lambda Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in lambda exonuclease involved in recognizing the 5'-phosphate at the ends of broken dsDNA. The preference of lambda exonuclease for a phosphate moiety at 5' dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5'-phosphate is due to the formation of inert enzyme-substrate complexes. By examining a lambda exonuclease mutant impaired in 5'-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of lambda exonuclease has been elucidated. We propose a model in which processivity of lambda exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.

Bacteriophage lambda↗
PubMed2006

Length-dependent degradation of single-stranded 3' ends by the Werner syndrome protein (WRN): implications for spatial orientation and coordinated 3' to 5' movement of its ATPase/helicase and exonuclease domains.

BACKGROUND: The cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Although WRN has been most prominently suggested to function in telomere maintenance, resolution of replication blockage and/or recombinational repair, its exact role in DNA metabolism remains unclear. WRN is the only human RecQ family member to possess both helicase and exonuclease activity, but the mechanistic relationship between these activities is unknown. In this study, model single-stranded and 3' overhang DNA substrates of varying length and structure were used to examine potential coordination between the ATPase/helicase and exonuclease activities of WRN. RESULT: Our results show that WRN can not only bind to but also catalyze the 3' to 5' degradation of single-stranded and 3' overhang DNA substrates, structures that were previously thought to be refractory to WRN exonuclease activity. The length of the single-stranded regions in these structures is a critical parameter in determining both the binding affinity and the level of exonuclease activity of WRN. Most importantly, specific nucleotide cofactors dramatically stimulate WRN exonuclease activity on these substrates, with conditions that permit ATP hydrolysis not only resulting in enhanced exonuclease activity but also altering its length dependence on these structures. Parallel experiments show that a deletion mutant containing only the WRN exonuclease domain lacks both this DNA length and nucleotide cofactor dependence, demonstrating that the interaction of the ATPase/helicase domain of WRN with the DNA substrate has a profound influence on exonuclease activity. CONCLUSION: Our results indicate that, under conditions that permit ATP hydrolysis, there is a dynamic and cooperative relationship between the distinct ATPase/helicase and exonuclease domains of WRN with regard to their orientation on DNA. Based on these results, models are proposed for the coordinated, unidirectional 3' to 5' movement of the helicase and exonuclease domains of WRN on DNA that should be informative for elucidating its function in genome maintenance.

Adenosine Triphosphatases↗
PubMed1989

The actions of Neurospora endo-exonuclease on double strand DNAs.

Neurospora crassa endo-exonuclease, an enzyme implicated in recombinational DNA repair, was found previously to have a distributive endonuclease activity with a high specificity for single strand DNA and a highly processive exonuclease activity. The activities of endo-exonuclease on double strand DNA substrates have been further explored. Endo-exonuclease was shown to have a low bona fide endonuclease activity with completely relaxed covalently closed circular DNA and made site-specific breaks in linear double strand DNA at a low frequency while simultaneously generating a relatively high level of single strand breaks (nicks) in the DNA. Sequencing at nicks induced by endo-exonuclease in pBR322 restriction fragments showed that the highest frequency of nicking occurred at the mid-points of two sites with the common sequence, p-AGCACT-OH. In addition, sequencing revealed less frequent nicking at identical or homologous hexanucleotide sequences in all other 54 cases examined where these sequences either straddled the break site itself or were within a few nucleotides on either side of the break site. The exonucleolytic action of endo-exonuclease on linear DNA showed about 100-fold preference for acting in the 5' to 3' direction. Removal of the 5'-terminal phosphates substantially reduced this activity, internal nicking, and the ability of endo-exonuclease to generate site-specific double strand breaks. On the other hand, nicking of the dephosphorylated double strand DNA with pancreatic DNase I stimulated the exonuclease activity by almost 5-fold, but no stimulation was observed when the DNA was nicked by Micrococcal nuclease. Thus, 5'-p termini either at double strand ends or at nicks in double strand DNA are entry points to the duplex from which endo-exonuclease diffuses linearly or "tracks" in the 5' to 3' direction to initiate its major endo- and exonucleolytic actions. The results are interpreted to show how it is possible for endo-exonuclease to generate single strand DNA for switching into a homologous duplex either at a nick or while remaining bound at a double strand break in the DNA. Such mechanisms are consistent with current models for recombinational double strand break repair in eukaryotes.

Base Composition↗
PubMed2003

Modulation of the 3'-->5'-exonuclease activity of human apurinic endonuclease (Ape1) by its 5'-incised Abasic DNA product.

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.

Catalysis↗
PubMed1996

Endo-exonuclease of human leukaemic cells: evidence for a role in apoptosis.

Inactive forms of endo-exonuclease, activated in vitro by treatment with trypsin, have been identified in human leukaemic CEM and MOLT-4 cells. They comprise over 95% of the total single-strand DNase activity in nuclei and are mainly bound to chromatin and the nuclear matrix. The activated enzyme had Mg2+(Mn2+)-dependent, Ca(2+)-stimulated activities with single- and double-strand DNAs and RNA (polyriboadenylic acid) and other properties characteristic of endo-exonucleases previously described. At least twice as much inactive endo-exonuclease has also been localised in extranuclear compartments of CEM and MOLT-4 cells, 85% bound to the membranes of the endoplasmic reticulum and 15% free in the cytosol. The soluble cytosolic trypsin-activatable endo-exonuclease was immunoprecipitated by antibodies raised independently to both Neurospora and monkey CV-1 cell endo-exonucleases. The free and bound enzymes of both nuclear and extranuclear compartments also cross-reacted on immunoblots with the antibody raised to Neurospora endo-exonuclease to reveal multiple polypeptides ranging in size from 18 to 145 kDa, many of which exhibited activity on DNA gels. The major species bound to the chromatin/matrix were in the 55-63 kDa range. Limited proteolysis of the large polypeptides to those of 18 to 46 kDa accompanied spontaneous chromatin DNA fragmentation to form DNA "ladders' in an isolated nuclei/cytosol system. When the leukaemic cells were treated in culture with either etoposide or podophyllotoxin to induce apoptosis, the largest polypeptides disappeared and smaller endo-exonuclease-related polypeptides of 18 to 46 kDa were detected in the nuclear extracts. The appearance of these polypeptides also correlated with extensive chromatin DNA fragmentation. In addition, there were correlations between the depletion of the major 55-63 kDa species bound to the membranes of the endoplasmic reticulum, depletion of the extranuclear trypsin-activatable activity and the onset and extent of chromatin DNA fragmentation in both cell lines. The extranuclear 55-63 kDa species may be precursors of the chromatin/matrix bound endo-exonuclease. The results indicate that endo-exonuclease plays a role in chromatin DNA degradation in mammalian cells during apoptosis.

Apoptosis↗
PubMed2002

The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis.

The 3'-->5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase gamma (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'-->5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.

DNA↗