Design of molecules that specifically recognize and cleave apurinic sites in DNA.
We have prepared a series of tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, a nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10(4) to 10(6) M-1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleave plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a beta-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable analog (tetrahydrofuran) of the abasic site for high field 1H NMR spectrometry and footprinting experiments. All results are consistent with a beta-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site recognition, phosphate binding and beta-elimination catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Berthet
N
N
L.E.D.S.S., URA CNRS 332, Université Joseph Fourier, Grenoble, France.
Boudali
A
A
Constant
J F
JF
Decout
J L
JL
Demeunynck
M
M
Fkyerat
A
A
Garcia
J
J
Laayoun
A
A
Michon
P
P
Lhomme
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D015195:Drug Design; D004720:Endonucleases / Q000378:metabolism; D066298:In Vitro Techniques; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; C029535:lysyl-tryptophyl-alpha-lysine; D004247:DNA; D004720:Endonucleases