Inhibition of the DNA unwinding and ATP hydrolysis activities of the bacteriophage T4 DDA helicase by a sequence specific DNA-protein complex.
The structural nature of helicase-substrate complexes in the unwinding mode is difficult to study due to their transient nature. We report here a simple method to freeze a DNA helicase at a specific position. The method employs a sequence-specific DNA protein complex as a "roadblock" to helicase movement. The feasibility of the approach is demonstrated by trapping the dda protein of bacteriophage T4 upstream of a GAL4-DNA complex. The presence of the trapped helicase is demonstrated directly by protection of a nearby restriction site and indirectly by the inability of the helicase to recycle rapidly to unwind an unmodified substrate. The half-life of this frozen complex is approximately two minutes under the conditions employed. These results suggest that further study of this novel complex will prove fruitful in elucidating the properties of a DNA helicase in its unwinding mode. As a case in point, it is shown that the dda protein ceases to hydrolyze ATP while stalled, suggesting that nucleotide triphosphate hydrolysis is coupled to translocation for this enzyme.
Maine
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IP
Department of Chemistry and Biochemistry, University of Texas at Austin 78712.
Kodadek
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T
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004265:DNA Helicases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4