Buffer composition mediates a switch between cooperative and independent binding of an initiator protein to DNA.
The regulation of many biological processes, including DNA replication, is frequently achieved by protein-protein interactions, as well as protein-DNA interactions. Multiple protein-binding sites are often involved. For example, the replication of plasmid R6K involves binding of the initiator protein pi to seven 22-bp direct repeats (DR) in the gamma origin of replication (gamma ori). A mutant protein pi S87N has been isolated, that in Tris.borate buffer (TB) binds cooperatively to seven DR, whereas wild-type (wt) pi binds independently [Filutowicz et al., Nucleic Acids Res. 22 (1994) 4211-4215]. Surprisingly, we found that wt pi can also bind cooperatively when Tris.acetate (TA), Tris.succinate or Tris.glutamate buffers are used instead of TB. The cooperative binding of the wt pi protein was also observed in the TB buffer at high concentrations of Na2EDTA. These results suggest that pi may be able to assume two functionally distinct conformations as a result of either mutation or buffer composition. Moreover, we found that the mode of pi binding is determined not by the composition of the buffer in which the reaction was assembled, but by the composition of the electrophoresis buffer. We discuss the general implications of these findings.
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Department of Bacteriology, University of Wisconsin-Madison 53706, USA.
York
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Filutowicz
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D000085:Acetates / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002021:Buffers; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004586:Electrophoresis; D018698:Glutamic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012091:Repetitive Sequences, Nucleic Acid; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid; D015534:Trans-Activators / Q000378:metabolism*; D014325:Tromethamine / Q000494:pharmacology
D000085:Acetates; D001426:Bacterial Proteins; D002021:Buffers; D004268:DNA-Binding Proteins; D010448:Peptide Initiation Factors; D013386:Succinates; D015534:Trans-Activators; C045264:replication initiator protein; D014325:Tromethamine; D018698:Glutamic Acid; D004533:Egtazic Acid; D004492:Edetic Acid; D019802:Succinic Acid; D004265:DNA Helicases