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Molecular Mechanism of DNA Deadenylation by the Neurological Disease Protein Aprataxin

2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês

10.1074/jbc.m807124200

1083-351X

Ulrich Rass, Ivan Ahel, Stephen C. West,

Mitochondrial Function and Pathology

The human neurological disease known as ataxia with oculomotor apraxia 1 is caused by mutations in the APTX gene that encodes Aprataxin (APTX) protein. APTX is a member of the histidine triad superfamily of nucleotide hydrolases and transferases but is distinct from other family members in that it acts upon DNA. The target of APTX is 5′-adenylates at DNA nicks or breaks that result from abortive DNA ligation reactions. In this work, we show that APTX acts as a nick sensor, which provides a mechanism to assess the adenylation status of unsealed nicks. When an adenylated nick is encountered by APTX, base pairing at the 5′ terminus of the nick is disrupted as the adenylate is accepted into the active site of the enzyme. Adenylate removal occurs by a two-step process that proceeds through a transient AMP-APTX covalent intermediate. These results pinpoint APTX as the first protein to adopt canonical histidine triad-type reaction chemistry for the repair of DNA. The human neurological disease known as ataxia with oculomotor apraxia 1 is caused by mutations in the APTX gene that encodes Aprataxin (APTX) protein. APTX is a member of the histidine triad superfamily of nucleotide hydrolases and transferases but is distinct from other family members in that it acts upon DNA. The target of APTX is 5′-adenylates at DNA nicks or breaks that result from abortive DNA ligation reactions. In this work, we show that APTX acts as a nick sensor, which provides a mechanism to assess the adenylation status of unsealed nicks. When an adenylated nick is encountered by APTX, base pairing at the 5′ terminus of the nick is disrupted as the adenylate is accepted into the active site of the enzyme. Adenylate removal occurs by a two-step process that proceeds through a transient AMP-APTX covalent intermediate. These results pinpoint APTX as the first protein to adopt canonical histidine triad-type reaction chemistry for the repair of DNA. Aprataxin is a novel DNA repair protein whose dysfunction causes the neurodegenerative disease ataxia with oculomotor apraxia 1 (AOA1), 2The abbreviations used are: AOA1, ataxia with oculomotor apraxia 1; HIT, histidine triad; SSB, single strand break; APTX, Aprataxin; FHA, forkhead-associated; ZF, zinc finger; HINT, histidine triad nucleotide-binding protein. which is characterized by cerebellar atrophy and sensorimotor neuropathy (1Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (337) Google Scholar, 2Moreira M.C. Barbot C. Tachi N. Kozuka N. Uchida E. Gibson T. Mendonca P. Costa M. Barros J. Yanagisawa T. Watanabe M. Ikeda Y. Aoki M. Nagata T. Coutinho P. Sequeiros J. Koenig M. Nat. Genet. 2001; 29: 189-193Crossref PubMed Scopus (381) Google Scholar, 3Rass U. Ahel I. West S.C. Cell. 2007; 130: 991-1004Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Cells derived from AOA1 patients are sensitive to DNA single strand break (SSB)-inducing agents such as hydrogen peroxide and methyl methanesulfonate (4Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair. 2004; 3: 1493-1502Crossref PubMed Scopus (165) Google Scholar, 5Gueven N. Becherel O.J. Kijas A.W. Chen P. Howe O. Rudolph J.H. Gatti R. Date H. Onodera O. Taucher-Scholz G. Lavin M.F. Hum. Mol. Genet. 2004; 13: 1081-1093Crossref PubMed Scopus (141) Google Scholar, 6Luo H. Chan D.W. Yang T. Rodriguez M. Chen B.P. Leng M. Mu J.J. Chen D. Songyang Z. Wang Y. Qin J. Mol. Cell. Biol. 2004; 24: 8356-8365Crossref PubMed Scopus (122) Google Scholar) and have been shown to accumulate SSBs after treatment with camptothecin and under conditions of oxidative stress (7Mosesso P. Piane M. Palitti F. Pepe G. Penna S. Chessa L. CMLS Cell. Mol. Life Sci. 2005; 62: 485-491Crossref PubMed Scopus (71) Google Scholar, 8Hirano M. Yamamoto A. Mori T. Lan L. Iwamoto T.A. Aoki M. Shimada K. Furiya Y. Kariya S. Asai H. Yasui A. Nishiwaki T. Imoto K. Kobayashi N. Kiriyama T. Nagata T. Konishi N. Itoyama Y. Ueno S. Ann. Neurol. 2007; 61: 162-174Crossref PubMed Scopus (64) Google Scholar). Human APTX is 342 amino acids in length and contains three functional domains: an N-terminal forkhead-associated (FHA) domain, a central histidine triad (HIT) domain, and a C-terminal zinc finger (ZF) domain (see Fig. 1A). The FHA domain mediates complex formation with XRCC1-DNA ligase IIIα by interaction with phosphorylated residues in XRCC1 (4Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair. 2004; 3: 1493-1502Crossref PubMed Scopus (165) Google Scholar, 5Gueven N. Becherel O.J. Kijas A.W. Chen P. Howe O. Rudolph J.H. Gatti R. Date H. Onodera O. Taucher-Scholz G. Lavin M.F. Hum. Mol. Genet. 2004; 13: 1081-1093Crossref PubMed Scopus (141) Google Scholar, 6Luo H. Chan D.W. Yang T. Rodriguez M. Chen B.P. Leng M. Mu J.J. Chen D. Songyang Z. Wang Y. Qin J. Mol. Cell. Biol. 2004; 24: 8356-8365Crossref PubMed Scopus (122) Google Scholar, 9Sano Y. Date H. Igarashi S. Onodera O. Oyake M. Takahashi T. Hayashi S. Morimatsu M. Takahashi H. Makifuchi T. Fukuhara N. Tsuji S. Ann. Neurol. 2004; 55: 241-249Crossref PubMed Scopus (73) Google Scholar), supporting a role for APTX in DNA single strand break repair. The FHA domain of APTX also interacts with phosphorylated XRCC4, however, thereby mediating complex formation with XRCC4-DNA ligase IV, suggestive of an additional role in double strand break repair (4Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair. 2004; 3: 1493-1502Crossref PubMed Scopus (165) Google Scholar). The actions of APTX are intimately linked to the mechanism by which DNA ligases promote break repair. Ligation occurs through a multistep process involving (i) the covalent binding of an adenylate (AMP) group to the active site lysine, (ii) transfer of the adenylate to the 5′-terminal phosphate group at the DNA break, and (iii) phosphodiester bond formation, which seals the DNA backbone and discharges the AMP moiety once again (10Ellenberger T. Tomkinson A.E. Annu. Rev. Biochem. 2008; 77: 313-338Crossref PubMed Scopus (258) Google Scholar). Recently, however, it has become apparent that 5′-DNA adenylates are not always resolved in the course of a ligation reaction. For example, it has been shown that human DNA ligase IIIα attempts futile ligation at "dirty" oxidative SSBs, which often have phosphates at both 3′ and 5′ termini, and aborts the reaction after adenylation of the DNA (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar). These dead-end adenylates appear to be the substrate for APTX as in vitro studies have shown that APTX can specifically remove 5′-adenylates, leading to the suggestion that APTX acts as a general proofreader for abortive DNA ligation reactions (3Rass U. Ahel I. West S.C. Cell. 2007; 130: 991-1004Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 12Rass U. Ahel I. West S.C. J. Biol. Chem. 2007; 282: 9469-9474Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 13Lavin M.F. Gueven N. Grattan-Smith P. DNA Repair. 2008; 7: 1061-1076Crossref PubMed Scopus (17) Google Scholar). This discovery of the DNA deadenylation reaction catalyzed by APTX provides a molecular rationale for AOA1 because abortive ligation events are inevitable in vivo given that oxidative damage causes many dirty breaks per cell each day. Normally these dead-end DNA adenylates would be resolved by APTX, but in AOA1 patients they may accumulate as a consequence of ATPX dysfunction, leading to impaired gene transcription and eventual cell death. The specific sensitivity of neuronal cells seen in AOA1 may be due to high levels of oxidative stress coupled with their postmitotic status, which might not permit alternative (replication-dependent) options for the removal and repair of DNA adenylates (3Rass U. Ahel I. West S.C. Cell. 2007; 130: 991-1004Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 14McKinnon P.J. Caldecott K.W. Annu. Rev. Genomics Hum. Genet. 2007; 8: 37-55Crossref PubMed Scopus (224) Google Scholar). APTX is a member of the HIT superfamily of proteins, which takes its name from the active site HϕHϕHϕϕ histidine triad sequence motif (where ϕ is a hydrophobic residue) (15Seraphin B. DNA Seq. 1992; 3: 177-179Crossref PubMed Scopus (82) Google Scholar). HIT proteins catalyze nucleotide transfer from a number of specific substrates to water (hydrolase activity) or a second substrate (transferase activity) (16Brenner C. Biochemistry. 2002; 41: 9003-9014Crossref PubMed Scopus (245) Google Scholar), and it is the HIT domain that gives APTX the capacity to deadenylate DNA (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar). In this study, we addressed the mechanism by which APTX interacts with DNA and resolves DNA adenylates. Significantly APTX is the only HIT superfamily member to act on DNA, and we provide evidence that the specificity for DNA adenylates is reflected in its HIT-ZF domain structure, which we propose is a defining and conserved feature of Aprataxin orthologs. We show that APTX possesses nick sensor activity, which is ideally suited to locate and repair 5′-DNA adenylates. The DNA deadenylation reaction involves two-step chemistry that proceeds through transient formation of a covalent AMP-enzyme intermediate. Extracts and Proteins—Human APTX cDNA was PCR-amplified from a HeLa cDNA library (Invitrogen) and cloned into pET41 using the SpeI and BamHI restriction sites. This plasmid expressed recombinant APTX fused to an N-terminal glutathione S-transferase tag and a C-terminal His8 tag. The QuikChange II site-directed mutagenesis kit (Stratagene) was used to create mutant versions of APTX. Arabidopsis thaliana total RNA was kindly provided by Dr. Charles White. cDNA corresponding to a transcript starting at nucleotide 1146 to the end of APTX (AT5G01310) was cloned into pET41a using the NcoI and BamHI restriction sites. The resulting protein, containing glutathione S-transferase/His6 tags at the N terminus, was expressed in Escherichia coli. The tags used here have been shown previously not to interfere with APTX activity (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar, 12Rass U. Ahel I. West S.C. J. Biol. Chem. 2007; 282: 9469-9474Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and were used to purify all recombinant APTXs to near homogeneity as described previously (12Rass U. Ahel I. West S.C. J. Biol. Chem. 2007; 282: 9469-9474Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Extracts from human lymphoblastoid cells, chicken DT40 cells, and yeast were prepared as described previously (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar), Caenorhabditis elegans extracts were a gift from Dr. Spencer Collis (17Polanowska J. Martin J.S. Garcia-Muse T. Petalcorin M.I. Boulton S.J. EMBO J. 2006; 25: 2178-2188Crossref PubMed Scopus (129) Google Scholar), and E. coli extracts were prepared by sonication of cells in a buffer containing 50 mm Tris-HCl, pH 8.0, 40 mm NaCl, 5 mm EDTA, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin, and 5% glycerol followed by centrifugation to remove cell debris. DNA Substrates—Synthetic DNA substrates were assembled from the following oligonucleotides: 1, 5′-ATTCCGATAGTGACTACA-3′; 2, 5′-CATATCCGTGTCGCCCT-3′P; 3, 5′-TGTAGTCACTATCGGAATGAGGGCGACACGGATATG-3′; 4, 5′-CATATCCGTGTCGCCCTCATTCCGATAGTGACTACA-3′; 1T, 5′-AGATTATCTTCGAGCTAC-3′; and 3T, 5′-GTAGCTCGAAGATAATCTGAGGGCGACACGGATATG-3′. All oligonucleotides were purchased from Sigma and purified by denaturing PAGE. Oligonucleotides were 5′-32P-labeled using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (GE Healthcare), and 5′-phosphorylation was brought to completion by addition of excess cold ATP. SSB substrates (2 μm) were adenylated by abortive ligation with 400 units of T4 DNA ligase (New England Biolabs) in a buffer containing 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10 mm dithiothreitol, and 2 mm ATP for 12 h. The adenylated strand (oligo 1) was purified by 15% denaturing PAGE and reannealed with oligos 2 and 3 to form adenylated SSB substrates (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar). The duplex substrate comprised oligo 3 with its complement (oligo 4). Neutral PAGE was used to purify all annealed DNA substrates before use. For the permanganate experiments, oligo 2 was annealed with oligos 1T and 3T. Adenylated ϕX174 DNA was prepared as described previously (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar). In brief, 95 nm ϕX174 replicative form I DNA (New England Biolabs) was treated with 10 μm H2O2, 0.1 mm FeCl3, 0.2 mm EDTA, 100 mm NaCl, and 1 mm NADH for 30 min at room temperature. Resulting oxidative DNA breaks were adenylated with [α-32P]ATP (GE Healthcare) by abortive ligation using T4 DNA ligase. DNA Deadenylation Reactions—Synthetic SSB substrates (25 nm) and adenylated ϕX174 replication form I DNA (6 nm) were used in 10-μl reactions containing protein extract or recombinant APTX as indicated. Unless stated otherwise, incubation was for 1 min at 37 °C in 50 mm Tris-HCl, pH 8.0, 40 mm NaCl, 5 mm EDTA, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin, and 5% glycerol. Reactions were scaled up for time course experiments. The deadenylation of synthetic SSB substrates was stopped by addition of formamide loading buffer and incubation at 95 °C for 3 min. Products were analyzed by 12% denaturing PAGE followed by autoradiography. Deadenylation reactions containing the ϕX174 DNA were stopped by addition of SDS (2% final concentration) and analyzed by 0.6% agarose gel electrophoresis or 10% SDS-PAGE followed by autoradiography. Data were quantified using a Storm 840 phosphorimaging system (GE Healthcare). DNase I Footprinting—Reactions (5 μl) contained the DNA substrate (25 nm) in DNase I buffer (10 mm Tris-HCl, pH 7.6, 2.5 mm MgCl2, and 0.5 mm CaCl2). After 15 min of incubation at 25 °C, 0.1 unit of DNase I (New England Biolabs) was added. DNase I digestion was stopped after 1 min by addition of formamide loading buffer and incubation at 95 °C for 3 min. Reaction products were analyzed by 12% denaturing PAGE followed by autoradiography. Chemical Cleavage of DNA—Reactions (10 μl) contained 25 nm DNA substrate, 50 mm Tris-HCl, pH 8.0, 30 mm NaCl, 5 mm EDTA, 1 mm dithiothreitol, 100 g/ml bovine serum albumin, 5% glycerol, and APTX as indicated. After preincubation for 20 min at 25 °C, KMnO4 was added to a final concentration of 5 mm. After 2 min β-mercaptoethanol was added to a final concentration of 1 m. After ethanol precipitation in the presence of 1 mg/ml glycogen, the DNA was resuspended in 1 m piperidine and incubated at 95 °C for 30 min. After ethanol precipitation, the DNA was resuspended in formamide loading buffer and analyzed by 12% denaturing PAGE followed by autoradiography. Molecular Architecture and Evolutionary Conservation of Aprataxin—Human APTX features a tripartite FHA-HIT-ZF domain structure (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar, 12Rass U. Ahel I. West S.C. J. Biol. Chem. 2007; 282: 9469-9474Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To determine whether the co-occurrence of a ZF with a HIT domain might indicate authentic Aprataxins, we performed data base searches to identify potential orthologs in virtually all sequenced eukaryotic genomes (Fig. 1A). One exception was found in C. elegans where, despite the presence of several HIT superfamily proteins, we were unable to detect the presence of a ZF in any of these proteins. HIT-ZF domain proteins were also absent from prokaryotes. When cell-free extracts prepared from a variety of organisms were incubated with a synthetic 5′-adenylated nicked DNA substrate with a one-nucleotide gap and phosphates at both the 3′ and the 5′ termini (Fig. 1B; this substrate mimics the product of an abortive ligation event that would occur at an SSB induced by oxidative DNA damage), we observed a perfect correlation between our HIT-ZF predictions and DNA deadenylation activity (Fig. 1C). The HIT-ZF domain proteins identified in plants are characterized by a more elaborate domain structure including a number of additional modules (Fig. 1A). To determine whether these are true Aprataxins, we cloned recombinant A. thaliana basic helix-loop-helix family protein AT5G01310.1 and tested its ability to catalyze DNA deadenylation. The recombinant protein spanned the macro-, HIT, and ZF domains of the protein and was proficient for DNA deadenylation in the presence or absence of Mg2+ ions, indicative of true APTX-like metal-independent DNA deadenylation activity (Fig. 1D, lanes 2 and 3) (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar). Taken together, these data show that APTX is functionally conserved in animals, fungi, and plants but not in prokaryotes and establish the combination of a HIT domain and a ZF domain as the structural hallmark of the Aprataxins. Nick Sensor Activity of Aprataxin—Previous studies have shown that APTX binds linear double-stranded DNA and nicked DNA substrates with similar affinity and that adenylated DNA is bound more avidly than non-adenylated substrates (12Rass U. Ahel I. West S.C. J. Biol. Chem. 2007; 282: 9469-9474Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Kijas A.W. Harris J.L. Harris J.M. Lavin M.F. J. Biol. Chem. 2006; 281: 13939-13948Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To define these interactions in more detail, we used DNase I protection assays to analyze complexes formed between APTX and DNA substrates containing 5′-adenylated nicks. To inhibit adenylate removal, it was necessary to use human APTX carrying the inactivating mutation H260A. Previous studies have shown that recombinant APTXH260A, with an N-terminal glutathione S-transferase tag and a C-terminal His8 tag, exhibits wild-type substrate binding properties (12Rass U. Ahel I. West S.C. J. Biol. Chem. 2007; 282: 9469-9474Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). APTXH260A was incubated with the adenylated SSB substrate (Fig. 2A; this contains a one-nucleotide gap and is composed of three oligonucleotides: an 18-mer with a 5′-adenylate (oligo 1), a 17-mer with a 3′-phosphate terminus at the 5′-side of the nick (oligo 2), and a continuous 36-mer (oligo 3)), and the resulting complexes were probed using DNase I. In the presence of APTX, regions of protection were observed over ∼12 nucleotides of oligo 1 and 14 nucleotides of oligo 3. In contrast, no significant changes were observed with oligo 2, indicating that Aprataxin binds asymmetrically to the DNA almost exclusively at the 3′-side of the adenylate (Fig. 2A and indicated schematically in Fig. 2B). When similar experiments were carried out with non-adenylated nicked DNA, we were surprised to find very similar protection patterns (Fig. 2, C and D) as again strong protection patterns were observed at the 3′-side of the nick. To ensure it was the nick that determined the specific positioning of APTX, we next analyzed complexes formed between APTXH260A and linear duplex DNA of the same length. In this case, we failed to see a clear protection pattern and instead observed weak protection over the entire DNA duplex (Fig. 2E; note that the apparent protection at the 5′ terminus of oligos 3 and 4 was due to the inhibition of multiple cleavage events in the presence of APTX). The nick sensor activity of APTX was disrupted when the ZF domain was inactivated by mutation of the critical zinc-coordinating residues Cys-319 and Cys-322 as the APTXH260A/C319A/C322A mutant protein failed to protect either adenylated or non-adenylated SSB substrates from DNase I digestion (data not shown). APTX Disrupts Watson-Crick Base Pairing at DNA Adenylates—Reasoning that to sample for and/or bind a 5′-AMP moiety, APTX might need to separate the Watson and Crick strands, we next determined whether the interaction of Aprataxin with 5′-adenylates results in base pair disruption. To do this, the formation of unpaired thymines close to the adenylate was monitored using potassium permanganate (KMnO4) as a specific probe. To enable these analyses, we incorporated thymine residues directly adjacent to the nick (T1) and at positions three (T2) and six (T3) nucleotides downstream of the nick on the continuous strand of the SSB substrate (oligo 3T), which was annealed with oligonucleotides 1T and 2 (Fig. 3B). Preliminary experiments using oligo 3T annealed with oligo 2 only (single-stranded/tailed duplex DNA) confirmed that thymine residues T1–T3 were readily accessible to the chemical probe when they were unpaired (Fig. 3A, lane 2). When oligo 3T was present in a non-adenylated SSB substrate, we observed that T1 remained sensitive to chemical cleavage in the absence of APTX, whereas all other thymine residues were protected (lane 5). These results indicate that the chemical probe retains access to the base adjacent to the nick possibly as a result of DNA breathing. In the presence of APTXH260A (lane 5) or wild-type APTX (lane 6), the degree of sensitivity at T1 was unchanged, and we did not find induced sensitivity at other thymine residues. These results show that although APTX binds the 3′-side of the nick (Fig. 2) binding per se does not lead to separation of the Watson and Crick strands (Fig. 3B, panel i). Parallel experiments were carried out with adenylated SSB substrates. Without APTX, we found that T1 was relatively insensitive to chemical cleavage (Fig. 3A, lane 8, and indicated schematically in Fig. 3B, panel ii), but the presence of APTXH260A resulted in a significant hypersensitivity of residue T1 to chemical attack (lane 9). In contrast, wild-type APTX failed to induce such a level of hypersensitivity as expected because the active protein will remove the adenylate under these reaction conditions (lane 10). These results show that the stable binding of APTX to its target lesion results in measurable base disruption to the nucleotide pair directly flanking the adenylated 5′-terminus of the nick (Fig. 3B, panel iii). No sensitivity was induced at T2 or T3, demonstrating that strand separation is limited to the immediate proximity of the adenylated base pair. These results demonstrate that the interaction of APTX with the DNA adenylate induces structural changes in the DNA that are a likely prerequisite for DNA deadenylation. A Covalent AMP-Enzyme Intermediate—Previously it was shown that DNA deadenylation by APTX results in the release of free AMP (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar), but the mechanism of adenylate hydrolysis remains unknown. The HIT hydrolases generally catalyze a two-step reaction, which includes (i) nucleotide transfer to the active site of the enzyme and (ii) hydrolysis of the resulting nucleotidylated enzyme intermediate (19Lima C.D. Klein M.G. Hendrickson W.A. Science. 1997; 278: 286-290Crossref PubMed Scopus (194) Google Scholar). To probe for a covalent AMP-APTX intermediate, we incubated APTX with plasmid DNA that contained 32P-labeled adenylates (Fig. 4A). This substrate was produced by treating plasmid DNA with hydrogen peroxide to produce dirty breaks and subsequent treatment of the damaged plasmid with DNA ligase in the presence of [α-32P]ATP (Fig. 4A) (11Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (294) Google Scholar). As expected, incubation with APTX resulted in rapid deadenylation of the DNA such that all radioactive label was lost (Fig. 4B, compare lanes 1 and 2). However, when aliquots of the deadenylation reaction were taken 0, 10, 20, 30, and 60 s after initiation of the reaction, and the products were resolved by SDS-PAGE, we observed the formation of a discrete 32P-labeled product that comigrated with recombinant APTX (Fig. 4C, lane 2). The formation of 32P-labeled APTX was even more apparent when reactions were carried out on ice (lane 6). These results demonstrate that DNA deadenylation proceeds through a short lived covalent AMP-APTX intermediate. Catalytic Role of Four Conserved Histidine Residues—Next we assessed the significance of a conserved network of active site hydrogen-bonding interactions for the DNA deadenylation reaction. Structural analysis of the HIT hydrolases HINT and fragile histidine triad protein has pinpointed two histidine residues within the histidine triad that make direct contacts with the α-phosphate of the nucleotide ligand (19Lima C.D. Klein M.G. Hendrickson W.A. Science. 1997; 278: 286-290Crossref PubMed Scopus (194) Google Scholar, 20Lima C.D. D'Amico K.L. Naday I. Rosenbaum G. Westbrook E.M. Hendrickson W.A. Structure (Lond.). 1997; 5: 763-774Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Lima C.D. Klein M.G. Weinstein I.B. Hendrickson W.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5357-5362Crossref PubMed Scopus (102) Google Scholar, 22Brenner C. Garrison P. Gilmour J. Peisach D. Ringe D. Petsko G.A. Lowenstein J.M. Nat. Struct. Biol. 1997; 4: 231-238Crossref PubMed Scopus (127) Google Scholar). These residues are stabilized by two further histidine residues, one situated within the histidine triad and one positioned upstream in the primary sequence. By analogy, we predict that APTX His-260 contacts the α-phosphate of the adenylate group with its N∊ atom and is stabilized by a hydrogen bond between the Nδ atom and the carbonyl oxygen atom of His-258, whereas His-262 contacts the α-phosphate and is stabilized by a hydrogen bond between its Nδ atom and the N∊ atom of the upstream residue His-201 (Fig. 5A). To test this model, we generated recombinant APTXH201A, APTXH258A, and APTXH262A mutant proteins and compared their activities with APTXH260A and the wild-type protein. First the activity of each protein was determined using the oligonucleotide-based adenylated SSB substrate (Fig. 5B). We observed substantial residual activity for APTXH258A (lane 4), weak activity for APTXH201A and APTXH262A (lanes 3 and 6), and no detectable activity with APTXH260A (lane 5). Similar catalytic activities were observed when we measured the loss of radiolabel from plasmid DNA that was adenylated with [32P]AMP (Fig. 5C). Interestingly when the products of the [32P]AMP plasmid reactions were analyzed by SDS-PAGE after 5 min, we observed a long lived covalent AMP-enzyme intermediate with APTXH258A indicative of impaired enzyme-adenylate hydrolysis (Fig. 5D, lane 3). The last position of the histidine triad is not occupied by a histidine but by a glutamine residue in the HIT nucleotide transferases (e.g. galactose-1-phosphate uridylyltransferase) (Fig. 5E). They also differ from the HIT nucleotide hydrolases by reacting with a second substrate rather than with water to resolve the nucleotidylated enzyme intermediate. To determine the effect of a glutamine residue at position 262 in APTX, we generated mutant APTXH262Q. In contrast to APTXH262A, this mutant showed significant residual DNA deadenylation activity without the need for a second substrate (Fig. 5F). Given that mutation of the four conserved histidine residues, His-201, His-258, His-260, and His-262, differentially affect the DNA deadenylation activity of APTX and that the H258A mutant exhibits a specific defect in the second reaction step, those mutants that retained substantial residual activity were examined more closely. We found that wild-type APTX deadenylated over 90% of the DNA substrate within the first 10 s of the reaction (Fig. 5G). In contrast, mutant APTXH262Q was defective in step 1 (90% DNA deadenylation was achieved only after 5 min of incubation), and yet the rate of release of the AMP from the AMP-APTXH262Q was not significantly affected. With APTXH258A, DNA deadenylation was only slightly slowed with 86% deadenylation taking place within 10 s. At the same time point, 30% of the 32P-labeled AMP was covalently linked to the protein compared with just 3% with the wild-type protein. Taken together, the results presented in Fig. 5 underline the importance of the hydrogen-bonding network that the four conserved histidines form among each other and with the substrate in HIT hydrolases. Consistent with the biochemical results, AOA1-related point mutations are found almost exclusively in the vicinity of the active site and particularly around the histidine triad and histidine residue 201 (Fig. 5A, panel ii) (3Rass U. Ahel I. West S.C. Cell. 2007; 130: 991-1004Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 23Caldecott K.W. Na