Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

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.     

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.     

Properties of and substrate determinants for the exonuclease activity of human apurinic endonuclease Ape1

Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5' to the damage site. This enzyme also functions in specific contexts to excise 3'-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively "minor" 3' to 5' exonuclease activity of Ape1 was found to contribute to the excision of certain 3'-mismatched nucleotides. In this study, I characterize more thoroughly the 3'-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3'-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5'-phosphate, compared to a 5'-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5'-abasic residue exerted an about tenfold attenuation on the 3' to 5' exonuclease efficiency of Ape1. A 3'-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase beta extension. Ape1 was also found to remove 3'-tyrosyl residues from 3'-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I-DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3'-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3'-flap-type endonuclease activity, again activating these substrates for polymerase beta extension.     

Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway

Most common point mutations occurring spontaneously or induced by ionizing radiation are C-->T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5' to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2'-deoxycytidine (5ohC) and alpha-anomeric 2'-deoxycytidine (alphadC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (NDelta31, NDelta35 and NDelta61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3'-->5' exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3'-->5' exonuclease activities generate a single-strand gap at the 5' side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo.     

AP endonuclease 1 has no biologically significant 3(')-->5(')-exonuclease activity

The 3(')-->5(')-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3(')-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3(')-->5(')-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3(')-->5(')-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3(')-3(') linkers. These do not require the -PuPu(Py)CGPyPy- hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-kappaB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-gamma secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.     

A 3'-5' exonuclease in human leukemia cells: implications for resistance to 1-beta -D-arabinofuranosylcytosine and 9-beta -D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate

A 3'-5' exonuclease that excises the nucleotide analogs 1-beta-d-arabinofuranosylcytosine monophosphate and 9-beta-d-arabinofuranosyl-2-fluoroadenine 5'-monophosphate incorporated at 3' ends of DNA was purified from the nuclei of: 1) primary human chronic lymphocytic leukemia cells, 2) primary and established human acute myeloblastic leukemia cells, and 3) lymphocytes obtained from healthy individuals. The activity of this nuclear exonuclease (exoN) is elevated approximately 6-fold in 1-beta-d-arabinofuranosylcytosine-resistant leukemia cells as compared with drug-sensitive cells, and it differs between two healthy individuals and among three leukemia patients. exoN is a 46-kDa monomer, requires 50 mm KCl and 1 mm magnesium for optimal activity, and shows a preference for single-stranded over duplex DNA. Its physical and enzymatic properties indicate that exoN is a previously uncharacterized enzyme whose activity may confer resistance to clinical nucleoside analogs in leukemia cells.     

Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.     

Characterization of the 3' exonuclease of Chlamydophila pneumoniae endonuclease IV on double-stranded DNA and the RNA strand of RNA/DNA hybrid

Endonuclease IV has AP endonuclease and 3'-repair diesterase activities. Here, we report Chlamydophila pneumoniae endonuclease IV (CpEndoIV) could hydrolyze the ds DNA and the RNA strand of RNA/DNA hybrid from the 3' end, yet the DNA strand of RNA/DNA hybrid was not the effective substrate of CpEndoIV. The optimal pH for 3' exonuclease on double-stranded (ds) DNA and RNA/DNA hybrids were both basic, but with some difference. The effect of divalent ions (Mg(2+), Ca(2+), Zn(2+), Cu(2+), Ni(2+), and Mn(2+)) on 3' exonuclease was different for both substrates. High concentration of NaCl inhibited 3' exonuclease on both substrates. For both substrates, the 3' exonuclease activity of CpEndoIV on matched and mismatched 3' end was comparable.     

Role of PCNA-dependent stimulation of 3′-phosphodiesterase and 3′–5′ exonuclease activities of human Ape2 in repair of oxidative DNA damage

Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.     

The 3'->5' exonuclease of Apn1 provides an alternative pathway to repair 7,8-dihydro-8-oxodeoxyguanosine in Saccharomyces cerevisiae

The 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), a major mutagenic DNA lesion, results either from direct oxidation of guanines or misincorporation of 8oxodGTP by DNA polymerases. At present, little is known about the mechanisms preventing the mutagenic action of 8oxodGTP in Saccharomyces cerevisiae. Herein, we report for the first time the identification of an alternative repair pathway for 8oxoG residues initiated by S. cerevisiae AP endonuclease Apn1, which is endowed with a robust progressive 3'-->5' exonuclease activity towards duplex DNA. We show that yeast cell extracts, as well as purified Apn1, excise misincorporated 8oxoG, providing a damage-cleansing function to DNA synthesis. Consistent with these results, deletion of both OGG1 encoding 8oxoG-DNA glycosylase and APN1 causes nearly 46-fold synergistic increase in the spontaneous mutation rate, and this enhanced mutagenesis is primarily due to G . C to T . A transversions. Expression of the bacterial 8oxodGTP triphosphotase MutT in the apn1Delta ogg1Delta mutant reduces the mutagenesis. Taken together, our results indicate that Apn1 is involved in an S. cerevisiae 8-oxoguanine-DNA glycosylase (Ogg1)-independent repair pathway for 8oxoG residues. Interestingly, the human major AP endonuclease, Ape1, also exhibits similar exonuclease activity towards 8oxoG residues, raising the possibility that this enzyme could participate in the prevention of mutations that would otherwise result from the incorporation of 8oxodGTP.     

The 3'-5' exonuclease of human DNA polymerase delta (pol delta) is regulated by pol delta accessory factors and deoxyribonucleoside triphosphates.

Human DNA polymerase delta (pol delta) is required for the synthesis of leading strand of simian virus 40 (SV40) DNA replication in vitro. Pol delta requires the accessory factors, proliferating cell nuclear antigen (PCNA), activator 1 (A1; also known as replication factor C [RF-C]), human single-stranded DNA binding protein (HSSB; also known as replication protein A [RP-A]) for the elongation of primed template DNA. Since pol delta has an associated 3'-5' exonuclease activity, the effect of pol delta accessory factors on the exonuclease activity was examined. The 3'-5' exonuclease activity was stimulated 8-10 fold by the addition of HSSB, and this stimulatory effect was preferential to HSSB since other SSBs from E. coli, T4 or adenovirus, had a little or no effect. The stimulatory effect of HSSB was markedly inhibited by the combined action of A1 and PCNA. Furthermore, the addition of deoxyribonucleoside triphosphates (dNTPs) completely abolished the effect of HSSB on the 3'-5' exonuclease activity even in the absence of pol delta accessory factors. These results suggest that accessory factors and dNTPs regulate both the polymerase and the 3'-5' exonuclease activities.     

SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA

Although the use of IFN-alpha in combination with ribavirin has improved the treatment efficacy of chronic hepatitis C virus (HCV) infection, 20-50% of patients still fail to eradicate the virus depending on the HCV genotype. Recently, overexpression of HCV core protein has been shown to inhibit IFN signaling and induce SOCS-3 expression. Aim of this study was to examine the putative role of SOCS proteins in IFN resistance. By Western blot analysis, a 4-fold induction of STAT-1/3 phosphorylation by IFN-alpha was observed in mock-transfected HepG2 clones. In contrast, IFN-induced STAT-1/3 phosphorylation was considerably downregulated by SOCS-1/3 overexpression. In mock-transfected cells, IFN-alpha induced 2',5'-OAS and myxovirus resistance A (MxA) promoter activity 40- to 80-fold and 10- to 35-fold, respectively, and this effect was abrogated in SOCS-1/3 overexpressing cells. As detected by Northern blot technique, IFN-alpha potently induced 2',5'-OAS and MxA mRNA expression in the control clones. Overexpression of SOCS-1 completely abolished both 2',5'-OAS and MxA mRNA expression, whereas SOCS-3 mainly inhibited 2',5'-OAS mRNA expression. Our results demonstrate that SOCS-1 and SOCS-3 proteins inhibit IFN-alpha-induced activation of the Jak-STAT pathway and expression of the antiviral proteins 2',5'-OAS and MxA. These data suggest a potential role of SOCS proteins in IFN resistance during antiviral treatment.     

A recombinant exonuclease III homologue of the thermophilic archaeon Methanothermobacter thermautotrophicus

AP endonucleases catalyse an important step in the base excision repair (BER) pathway by incising the phosphodiester backbone of damaged DNA immediately 5' to an abasic site. Here, we report the cloning and expression of the 774 bp Mth0212 gene from the thermophilic archaeon Methanothermobacter thermautotrophicus, which codes for a putative AP endonuclease. The 30.3 kDa protein shares 30% sequence identity with exonuclease III (ExoIII) of Escherichia coli and 40% sequence identity with the human AP endonuclease Ape1. The gene was amplified from a culture sample and cloned into an expression vector. Using an E. coli host, the thermophilic protein could be produced and purified. Characterization of the enzymatic activity revealed strong binding and Mg2+-dependent nicking activity on undamaged double-stranded (ds) DNA at low ionic strength, even at temperatures below the optimum growth temperature of M. thermautotrophicus (65 degrees C). Additionally, a much faster nicking activity on AP site containing DNA was demonstrated. Unspecific incision of undamaged ds DNA was nearly inhibited at KCl concentration of approximately 0.5 M, whereas incision at AP sites was still complete at such salt concentrations. Nicked DNA was further degraded at temperatures above 50 degrees C, probably by an exonucleolytic activity of the enzyme, which was also found on recessed 3' ends of linearized ds DNA. The enzyme was active at temperatures up to 70 degrees C and, using circular dichroism spectroscopy, shown to denature at temperatures approaching 80 degrees C. Considering the high intracellular potassium ion concentration in M. thermautotrophicus, our results suggest that the characterized thermophilic enzyme acts as an AP endonuclease in vivo with similar activities as Ape1.     

Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease

The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates.     

A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'- 5' exonuclease and 5'-protruding strand endonuclease activities

Werner syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype, genomic instability, and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1432-amino acid helicase/exonuclease (hWRN) have been shown to be responsible for the development of this disease. We have cloned, overexpressed, and purified a minimal, 171-amino acid fragment of hWRN that functions as an exonuclease. This fragment, encompassing residues 70-240 of hWRN (hWRN-N(70-240)), exhibits the same level of 3'-5' exonuclease activity as the previously described exonuclease fragment encompassing residues 1-333 of the full-length protein. The fragment also contains a 5'-protruding DNA strand endonuclease activity at a single-strand-double-strand DNA junction and within single-stranded DNA, as well as a 3'-5' exonuclease activity on single-stranded DNA. We find hWRN-N(70-240) is in a trimer-hexamer equilibrium in the absence of DNA when examined by gel filtration chromatography and atomic force microscopy. Upon addition of DNA substrate, hWRN-N(70-240) forms a hexamer and interacts with the recessed 3'-end of the DNA. Moreover, we find that the interaction of hWRN-N(70-240) with the replication protein PCNA also causes this minimal, 171-amino acid exonuclease region to form a hexamer. Thus, the active form of this minimal exonuclease fragment of human WRN appears to be a hexamer. The implications these results have on our understanding of hWRN's roles in DNA replication and repair are discussed.     

The crystal structure of TREX1 explains the 3' nucleotide specificity and reveals a polyproline II helix for protein partnering

The TREX1 enzyme processes DNA ends as the major 3' --> 5' exonuclease activity in human cells. Mutations in the TREX1 gene are an underlying cause of the neurological brain disease Aicardi-Goutières syndrome implicating TREX1 dysfunction in an aberrant immune response. TREX1 action during apoptosis likely prevents autoimmune reaction to DNA that would otherwise persist. To understand the impact of TREX1 mutations identified in patients with Aicardi-Goutières syndrome on structure and activity we determined the x-ray crystal structure of the dimeric mouse TREX1 protein in substrate and product complexes containing single-stranded DNA and deoxyadenosine monophosphate, respectively. The structures show the specific interactions between the bound nucleotides and the residues lining the binding pocket of the 3' terminal nucleotide within the enzyme active site that account for specificity, and provide the molecular basis for understanding mutations that lead to disease. Three mutant forms of TREX1 protein identified in patients with Aicardi-Goutières syndrome were prepared and the measured activities show that these specific mutations reduce enzyme activity by 4-35,000-fold. The structure also reveals an 8-amino acid polyproline II helix within the TREX1 enzyme that suggests a mechanism for interactions of this exonuclease with other protein complexes.     

The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5′-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2′-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2′-deoxyuridine, alpha-2′-deoxyadenosine and alpha-thymidine adducts, generating 3′-hydroxyl and 5′-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl₂ concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed.