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Arginine Residues 47 and 70 of Human Flap Endonuclease-1 Are Involved in DNA Substrate Interactions and Cleavage Site Determination
- Source :
- Journal of Biological Chemistry. 277:24659-24666
- Publication Year :
- 2002
- Publisher :
- Elsevier BV, 2002.
-
Abstract
- Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg(2+) on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant.
- Subjects :
- DNA Replication
Models, Molecular
Exonuclease
DNA Repair
Flap Endonucleases
Protein Conformation
DNA repair
Flap structure-specific endonuclease 1
Biochemistry
Substrate Specificity
chemistry.chemical_compound
Catalytic Domain
Humans
Amino Acid Sequence
Flap endonuclease
Molecular Biology
DNA Primers
Nuclease
Binding Sites
Endodeoxyribonucleases
Base Sequence
biology
DNA replication
DNA
Cell Biology
Recombinant Proteins
Kinetics
chemistry
Mutagenesis, Site-Directed
biology.protein
Flap endonuclease activity
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 277
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....785b139c017e8bba9dc9df26aacf5d7a