title: Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease.
abstract: 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 AP-site. 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.
authors.affiliation: Department of Biology, Northeastern University, Boston MA 02115, USA.
authors.forename: Luisa F
authors.initials: LF
authors.lastname: Melo
authors.forename: Sophia T
authors.initials: ST
authors.lastname: Mundle
authors.forename: Michael H
authors.initials: MH
authors.lastname: Fattal
authors.forename: N Edel
authors.initials: NE
authors.lastname: O'Regan
authors.forename: Phyllis R
authors.initials: PR
authors.lastname: Strauss
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007700:Kinetics; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014443:Tyrosine; D004247:DNA; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase