Nucleic Acids Research, 1993, Vol. 21, No. 18 4281-4287
© 1993
MOLECULAR BIOLOGY |
(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting
Department of Micorbiology Austin, TX 78712-1074, USA 1Drug Dynamics Institute, College of Pharmacy, University of Texas at Austin Austin, TX 78712-1074, USA
*To whom correspondence should be addressed
Received May 27, 1993. Revised July 23, 1993. Accepted July 23, 1993.
Phage Mu transposase (A-protein) Is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites Interaction of a protein monomer with DNA Is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90°. To probe the directionality and locus of these A-protein-lnduced bends, we have used the, antitumor antibiotic (+ )-CC-1065 as a structural probe. As a consequence of binding within the minor groove, ( + )-CC-1065 is able to alky late N3 of adenine in a sequence selective manner. This selectivity Is partially determined by conformatlonal flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure In which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-proteln - DNA complex or the (+ )-CC-1065 alkylatlon sites on DNA (aft site L3), we have demonstrated that of the three minor grooves implicated in the Interaction with A-proteln, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protelninduced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The Intervening minor groove between these two peripheral sites is protected from ( + )-CC-1065 alkylatlon. The results are discussed In reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of ( + )-CC- 1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.
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