Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.
Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
D J
DJ
Hosfield
G
G
Frank
Y
Y
Weng
J A
JA
Tainer
B
B
Shen
D000143:Acids; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics*; D019609:Archaeoglobus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D019605:Euryarchaeota / Q000201:enzymology* / Q000235:genetics; D045585:Flap Endonucleases; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000143:Acids; D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human