Substrate-specific inhibition of RecQ helicase.
The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (K(i)) for NMM inhibition of G4 DNA unwinding is 1.7 microM, approximately two orders of magnitude below the K(i) for inhibition of duplex DNA unwinding (>100 microM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.
Wu
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Department of Molecular Biophysics and Biochemistry and Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8024, USA.
Maizels
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D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D020128:Inhibitory Concentration 50; D008652:Mesoporphyrins / Q000737:chemistry / Q000494:pharmacology*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011166:Porphyrins / Q000737:chemistry / Q000494:pharmacology; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; D008652:Mesoporphyrins; D011166:Porphyrins; D011994:Recombinant Proteins; C065420:N-methylmesoporphyrin IX; C022571:adenosine 5'-O-(3-thiotriphosphate); C021096:tetra(4-N-methylpyridyl)porphine; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases