Interactions between branched DNAs and peptide inhibitors of DNA repair.
The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K(d) = 14 nM) and approximately 4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides' mechanism of action will further increase their utility.
Kepple
Kevin V
KV
Center for Microbial Sciences and Department of Biology, San Diego State University, San Diego, CA 92182, USA.
Patel
Namita
N
Salamon
Peter
P
Segall
Anca M
AM
D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004265:DNA Helicases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004260:DNA Repair / Q000187:drug effects*; D004261:DNA Replication; D045566:DNA, Cruciform / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding
D045566:DNA, Cruciform; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009842:Oligopeptides; C071446:RecG protein, E coli; D004247:DNA; D004265:DNA Helicases; D004229:Dithiothreitol