Endonuclease III interactions with DNA substrates. 1. Binding and footprinting studies with oligonucleotides containing a reduced apyrimidinic site.
The binding of endonuclease III from Escherichia coli to damaged DNA has been studied using gel shift and footprinting assays. Oligonucleotides containing a reduced apyrimidinic (AP) site were used since reduction of the AP site blocks the beta-elimination reaction catalyzed by the enzyme and yields a noncleavable substrate. The Kobs for a 13-mer carrying a centrally located reduced AP site is (2 x 10(6)-(2 x 10(7) M-1, while the Kobs for a 13-mer with no damage is (4.5 x 10(3)-(3.2 x 10(4) M-1 (approximately a 500-fold difference). Larger oligonucleotides would not enter a gel when endonuclease III was bound so that binding constants to oligonucleotides longer than 13 base pairs could not be determined directly. Competition assays suggest that the Kobs measured for both damaged and undamaged 13-mers is a minimum value and that the Kobs for larger oligonucleotides could be an order of magnitude greater. Fluorescence quenching on related 19-mers yielded a specific binding constant for the 19-mer carrying a centrally located reduced AP site for 4 x 10(7) M-1 and a nonspecific binding constant to an undamaged 19-mer of approximately 10(5) M-1 [Xing, D., Dorr, R., Cunningham, R. P., & Scholes, C. P. (1995) Biochemistry 34, 2537-2544]. Several footprinting reagents were used to determine the size and location of the endonuclease III binding site on damaged oligonucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Handley
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S
Department of Biological Sciences, University at Albany, State University of New York 12222.
Scholes
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CP
Cunningham
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D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry; D013379:Substrate Specificity
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli