Interaction of human apurinic endonuclease and DNA polymerase in the base excision repair pathway (original) (raw)
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Mutation research, 2000
DNA damage occurs unceasingly in all cells. Spontaneous DNA base loss, as well as the removal of damaged DNA bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinic/apyrimidinic (AP) sites. AP sites are the central intermediate in DNA base excision repair (BER) and must be processed by 5' AP endonucleases. These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5' of, AP sites to create a free 3'-OH end for DNA polymerase repair synthesis. In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these organisms and are homologous to the Escherichia coli enzymes Exonuclease III (Exo III) and Endonuclease IV (Endo IV), respectively. These enzymes represent both of the conserved 5' AP endonuclease enzyme families that exist in biology. Crystal structures of APE1 and Endo IV, bo...
DNA damage occurs unceasingly in all cells. Spontaneous DNA base loss, as well as the removal of damaged DNA Ž. bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinicrapyrimidinic AP sites. Ž. X AP sites are the central intermediate in DNA base excision repair BER and must be processed by 5 AP endonucleases. These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5 X of, AP sites to create a free 3 X-OH end for DNA polymerase repair synthesis. In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these Ž. Ž. organisms and are homologous to the Escherichia coli enzymes Exonuclease III Exo III and Endonuclease IV Endo IV , respectively. These enzymes represent both of the conserved 5 X AP endonuclease enzyme families that exist in biology. Crystal structures of APE1 and Endo IV, both bound to AP site-containing DNA reveal how abasic sites are recognized and the DNA phosphodiester backbone cleaved by these two structurally unrelated enzymes with distinct chemical mechanisms. Both enzymes orient the AP–DNA via positively charged complementary surfaces and insert loops into the DNA base stack, bending and kinking the DNA to promote flipping of the AP site into a sequestered enzyme pocket that excludes undamaged nucleotides. Each enzyme–DNA complex exhibits distinctly different DNA conformations, which may impact upon the biological functions of each enzyme within BER signal-transduction pathways. q
Journal of Biological Chemistry, 2003
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 335-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 . 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 . 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  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 .
Action of human apurinic endonuclease (Ape1) on C1′-oxidized deoxyribose damage in DNA
DNA Repair, 2003
Oxidative damage to DNA includes diverse lesions in the sugar-phosphate backbone. The chemical "nuclease" bis(1,10-phenanthroline)copper complex [(OP) 2 Cu] is believed to generate a mixture of direct oxidative strand breaks and C1-oxidized abasic sites (2-deoxyribonolactone; dL). We found that, under our conditions, the lesions produced by (OP) 2 Cu (50 M) in synthetic duplex DNA were predominantly dL, accompanied by ∼30% direct strand breaks with 3-phosphates. For enzymatic studies, (OP) 2 Cu was used to introduce damage with limited sequence-selectivity, while photolysis of a site-specific 2-deoxyuridine-1-t-butyl ketone generated dL at a defined position. The results showed that Ape1, the major human abasic endonuclease, catalyzed 5-incision of dL sites, but acted at least 10-fold less effectively to remove the 3-phosphates at direct strand breaks. Kinetic analysis of Ape1 incision using the site-specific dL substrate revealed the same k cat for dL and regular (glycosylase-generated) abasic sites, but with K m approximately five-fold higher for dL substrate. The efficiency of Ape1 acting on dL, and the abundance of this enzyme in vivo, indicate that dL sites in vivo would be rapidly processed by the endonuclease. The recent observation that Ape1-cleaved dL sites can covalently trap DNA polymerase  during the abasic excision process suggests that efficient incision of dL by Ape1 may potentiate further problems in DNA repair.
Journal of Biological …, 2004
Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5-endonuclease activity and 3-"endtrimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex ؉/؊ ). Our data indicate that Apex ؉/؊ mice are indeed APE-haploinsufficient, as exhibited by a 40 -50% reduction (p < 0.05) in APE mRNA, protein, and 5-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase  and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase -dependent BER response to cellular stress.
Characterization of the DNA polymerase requirement of human base excision repair
Nucleic Acids Research, 1996
Base excision repair is one of the major mechanisms by which cells correct damaged DNA. We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template. In this report, we demonstrate the fractionation of a human cell extract into two required components. One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase β (Polβ). Purified, recombinant Polβ efficiently substituted for this fraction. Escherichia coli PolI, mammalian Polδ and to a lesser extent Polα and ε also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair in human cell extracts. A neutralizing antibody to Polβ, which inhibited repair synthesis catalyzed by pure Polβ by ∼90%, only suppressed repair in crude extracts by a maximum of ∼70%. An inhibitor of Polβ, ddCTP, decreased base excision repair in crude extracts by ∼50%, whereas the Polα/δ/ε inhibitor, aphidicolin, reduced the reaction by ∼20%. A combination of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Polβ is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.
Proceedings of the National Academy of Sciences, 2006
The multifunctional DNA repair enzymes apurinic͞apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3-blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5 of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3-repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing ␣-anomeric 2-deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions. apurinic͞apyrimidinic endonuclease ͉ oxidative DNA damage ͉ tert-butyl hydroperoxide ͉ 3Ј-blocking groups ͉ ␣-anomeric 2Ј-deoxyadenosine
The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA
Mutation Research/DNA Repair, 2001
DNA continuously suffers the loss of its constituent bases, and thereby, a loss of potentially vital genetic information. Sites of missing bases-termed abasic or apurinic/apyrimidinic (AP) sites-form spontaneously, through damage-induced hydrolytic base release, or by enzyme-catalyzed removal of modified or mismatched bases during base excision repair (BER). In this review, we discuss the structural and biological consequences of abasic lesions in DNA, as well as the multiple repair pathways for such damage, while emphasizing the mechanistic operation of the multi-functional human abasic endonuclease APE1 (or REF-1) and its potential relationship to disease.
The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway
Nucleic Acids Research, 2004
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 modi®ed 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-speci®c 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 alphathymidine adducts, generating 3¢-hydroxyl and 5¢phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly ef®cient. The substrate speci®city and protein conformation of Ape1 is modulated by MgCl 2 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.
Journal of Biological Chemistry, 1998
Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase , which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3-phosphoglycolates 25-fold more slowly than C-4keto-C-1-aldehydes. DNA polymerase  excised the 5terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase -mediated excision of 5-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3-phosphoglycolate esters.