Facile interconversion of duplex structures formed by copolymers of d(CG).
Correlations between DNA sequence and reactivity have often been drawn with an implicit or explicit connection to duplex structure. An in vitro model using oligonucleotides of defined sequences has been developed to characterize a potential source of the hypersensitivity that naturally occurring regions of redundant sequence exhibit with many nucleases. S-1 nuclease was used here to diagnose the unusual hybridization of copolymeric DNA, d(CG)6, and related oligomers, through product and kinetic analysis. Fully complementary but redundant sequences reacted with this enzyme almost an order of magnitude faster than did heterogeneous fragments of DNA. Hydrolysis products of the copolymers indicated that conformations with unpaired termini were the sole substrates under these studies, and only a facile equilibrium between aligned and extended structures was required to explain the heightened reactivity of this DNA. For example, d(CG)6 was converted to d(CG)5 and d(CG)4 whereas d(CG)4C was initially processed to an octamer and then only later to a hexamer. Catalysis by S-1 exhibited no other substrate or product specificity; even the disordered bases in the loop region of a hairpin structure, d(CG)3T4(CG)3, did not provide sites of enhanced enzyme action. The rate of DNA consumption under standard conditions was proportional to the expected concentration of overhanging sequences rather than the absolute amount of DNA present. All initial attempts to saturate enzyme activity failed, and therefore, the rate of substrate formation through strand slippage was always faster than the catalytic depletion of unpaired bases. Only a low-energy transition state(s) must then separate the various hybridized species since this structural equilibration proceeded readily under conditions of 10 mM potassium phosphate, pH 7, 100 mM NaCl, and 22 degrees C.
Rokita
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Department of Chemistry, State University of New York, Stony Brook 11794.
Romero-Fredes
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D001483:Base Sequence; D004247:DNA; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides; D015719:Single-Strand Specific DNA and RNA Endonucleases
D009841:Oligonucleotides; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases