Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.
Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg(2+) and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.
Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Susan E
SE
Tsutakawa
Scripps Research Institute, La Jolla, California 92037.
David S
DS
Shin
Scripps Research Institute, La Jolla, California 92037.
Clifford D
CD
Mol
University of Kentucky, Lexington, Kentucky 40536; University of Texas Medical Branch, Galveston, Texas 77555.
Tadahide
T
Izumi
Scripps Research Institute, La Jolla, California 92037.
Andrew S
AS
Arvai
University of Texas Medical Branch, Galveston, Texas 77555.
Anil K
AK
Mantha
University of Texas Medical Branch, Galveston, Texas 77555.
Bartosz
B
Szczesny
Georgia State University, Atlanta, Georgia 30302.
Ivaylo N
IN
Ivanov
Scripps Research Institute, La Jolla, California 92037.
David J
DJ
Hosfield
Georgia State University, Atlanta, Georgia 30302.
Buddhadev
B
Maiti
Scripps Research Institute, La Jolla, California 92037.
Mike E
ME
Pique
Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Kenneth A
KA
Frankel
Lawrence Berkeley National Laboratory, Berkeley, California 94720; Scripps Research Institute, La Jolla, California 92037; Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
Kenichi
K
Hitomi
University of Albany, State University of New York, Albany, New York 12222.
Richard P
RP
Cunningham
University of Texas Medical Branch, Galveston, Texas 77555.
Sankar
S
Mitra
Lawrence Berkeley National Laboratory, Berkeley, California 94720; Scripps Research Institute, La Jolla, California 92037. Electronic address: JATainer@lbl.gov.
John A
JA
Tainer
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000737:chemistry*; D004926:Escherichia coli; D006801:Humans; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase