Modeling of flap endonuclease interactions with DNA substrate

Hatim T. Allawi, Michael W. Kaiser, Alexey V. Onufriev, Wu Po Ma, Andrew E. Brogaard, David A. Case, Bruce P. Neri, Victor I. Lyamichev

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

Structure-specific 5′ nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5′ flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5′ flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2′-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2′-O-methyl-modified substrates. To further refine the model, 5′ flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's βAB region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5′ flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3′ end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5′ nucleases.

Original languageEnglish (US)
Pages (from-to)537-554
Number of pages18
JournalJournal of molecular biology
Volume328
Issue number3
DOIs
StatePublished - May 2 2003
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • 2′-O-methyl
  • FEN1
  • Methylphosphonate
  • Point mutation
  • Structure-specific 5′ nuclease

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