(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: Overcoming limitations of hydroxyl-radical footprinting

Zhi Ming Ding, Rasika M. Harshey, Laurence H. Hurley

Research output: Research - peer-reviewArticle

  • 18 Citations

Abstract

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-induced 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 alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational 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-protein - DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, 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-protein-induced 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 alkylation. 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.

LanguageEnglish (US)
Pages4281-4287
Number of pages7
JournalNucleic Acids Research
Volume21
Issue number18
StatePublished - Sep 11 1993
Externally publishedYes

Fingerprint

CC 1065
Hydroxyl Radical
DNA
mu transposase
Probe
Protein
Proteins
Minor
Pharmaceutical Preparations
Drugs
Interaction
Alkylation
Experiments
Locus
Experiment
Bacteriophages
DNA sequences
Adenine
Genes
Monomers

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA : Overcoming limitations of hydroxyl-radical footprinting. / Ding, Zhi Ming; Harshey, Rasika M.; Hurley, Laurence H.

In: Nucleic Acids Research, Vol. 21, No. 18, 11.09.1993, p. 4281-4287.

Research output: Research - peer-reviewArticle

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