DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination

Mol, Clifford D.; Izumi, Tohru; Mitra, Sankar; Tainer, John A.

doi:10.1038/35000249

Cited by 686 publications

(939 citation statements)

References 30 publications

(1 reference statement)

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“…Tyr 128 and Tyr 269 contribute to endonuclease activity primarily through recognition and binding of DNA. Both are present outside the active site but make contact with substrate DNA (33). On the other hand, Tyr 171 contributes to enzymatic function through direct involvement in catalysis as well as binding and recognition of DNA.…”

Section: Discussionmentioning

confidence: 99%

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Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

et al. 2007

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AP endonuclease (AP endo), a key enzyme in repair of abasic sites in DNA, makes a single nick 5′ to the phosphodeoxyribose of an abasic site (AP-site). We recently proposed a novel mechanism, whereby the enzyme uses a key tyrosine (Tyr 171 ) to directly attack the scissile phosphate of the APsite. We showed that loss of the tyrosyl hydroxyl from Tyr 171 resulted in dramatic diminution in enzymatic efficiency. Here we extend the previous work to compare binding/recognition of AP endo to oligomeric DNA with and without an AP-site by wild type enzyme and several tyrosine mutants including Tyr 128 , Tyr 171 and Tyr 269 . We used single turnover and electrophoretic mobility shift assays. As expected, binding to DNA with an AP-site is more efficient than binding to DNA without one. Unlike catalytic cleavage by AP endo, which requires both hydroxyl and aromatic moieties of Tyr 171 , the ability to bind DNA efficiently without an AP-site is independent of an aromatic moiety at position 171. However, the ability to discriminate efficiently between DNA with and without an AP-site requires tyrosine at position 171. Thus, AP endo requires a tyrosine at the active site for the properties that enable it to behave as an efficient, processive endonuclease.

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Section: Discussionmentioning

confidence: 99%

“…These residues interact with AP-site containing oligonucleotide just upstream and downstream of the AP-site in the co-crystal structure (33). Mutation of either of these residues to alanine results in loss of one-two orders of magnitude in enzymatic efficiency (35 (35)).…”

Section: K D Values Determined By St/ssmentioning

confidence: 99%

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

et al. 2007

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“…The demonstration that the active site geometry of human APE1 appears optimal to retain the cleaved DNA product suggested that this enzyme acts to coordinate BER and led Tainer and colleagues to propose that BER results from the orderly transfer of unstable DNA intermediates between enzymes involved in this pathway. 95 According to this model, the unique structure of each DNA intermediate is recognized specifically by the enzyme that is required for the subsequent step of BER. For example, APE1 would "hand off " a cleaved intermediate to Pol β, which would then remove the dRP moiety and fill in the gap and relay the nicked intermediate to the XRCC1-DNA LIGIIIα complex for ligation.…”

Section: Linkage Between Aberrant Ber and Cancermentioning

confidence: 99%

Is Base Excision Repair a Tumor Suppressor Mechanism?

Sweasy¹,

Lang²,

DiMaio³

2006

Cell Cycle

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The base excision repair pathway is critical for the removal of oxidized and methylated bases from the DNA. Much of this DNA damage arises endogenously, as a result of oxygen metabolism. Several proteins including DNA glycosylases, the APE1 endonuclease, DNA polymerase β and DNA ligase, act in a highly regulated and coordinated manner during base excision repair to excise the base adducts from the DNA and restore the normal DNA sequence. Both germline and tumor-associated variants of genes encoding these proteins have been identified in humans. In many cases, the protein variant has been shown to have properties that could contribute to the development of cancer, suggesting that base excision repair acts as a tumor suppressor mechanism in humans. Limited epidemiological studies are consistent with this view. Our review of the literature indicates that additional laboratory and epidemiological studies of the role of base excision repair in cancer etiology is warranted.

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“…This enzyme recognizes AP sites in DNA and cleaves immediately 5 ¶ to the lesion, generating a DNA strand break with a priming 3 ¶-hydroxyl group and a 5 ¶-abasic residue. Prior biochemical and structural biology studies have indicated a model (known as ''passing the baton'') whereby human APE1 cooperates with the next protein in BER (i.e., DNA polymerase h) to coordinately execute the next steps of the corrective response (10). In addition to AP sites, APE1 exhibits repair activity on a variety of modified (nonconventional) 3 ¶-DNA termini, including, but not limited to, phosphates and phosphogycolyates (ref.…”

Section: Introductionmentioning

confidence: 99%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

107

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Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3 ¶ damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu 96 (changed to Gln) and Asp 210 (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue B-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.

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DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination

Cited by 686 publications

References 30 publications

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

Is Base Excision Repair a Tumor Suppressor Mechanism?

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

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