The use of Taq DNA polymerase to determine the sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II), acridine-tethered platinum(II) diammine complexes or two analogues.
cis-Diamminedichloroplatinum(II) (cisplatin) forms adducts with DNA. The sequence specificity of formation of cisplatin adducts with plasmid DNA was investigated using Taq DNA polymerase. This procedure involved the extension of an oligonucleotide primer by Taq DNA polymerase up to the cisplatin adduct. Using thermal cycling, this process is repeated many times in order to amplify the signal. The products of this linear amplification can then be examined on DNA sequencing gels, and the sequence specificity of cisplatin adduct formation can be determined to the exact base pair. In the pUC8 plasmid, the sequences that produced the most intense damage sites (as determined by densitometry) were runs of two or more Gs. Adducts could also be detected at GA, AG, and GC dinucleotides. Four other cisplatin analogues were also tested in the system. Two of these analogues contained an attached intercalating chromophore, and the strong damage with these compounds was similar to that found for cisplatin, but the medium and weak damage tended to be different. Weak damage was also detected with trans-diamminedichloroplatinum(II). With this compound, a large number of the damage sites were at the CG dinucleotide. This technique represents a simple, accurate, and quick method for determining the sequence specificity of damage for a cisplatin analogue in any DNA sequence.
Murray
V
V
School of Biochemistry, University of New South Wales, Kensington, Australia.
Motyka
H
H
England
P R
PR
Wickham
G
G
Lee
H H
HH
Denny
W A
WA
McFadyen
W D
WD
D001345:Autoradiography; D001483:Base Sequence; D002945:Cisplatin / Q000633:toxicity*; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009944:Organoplatinum Compounds / Q000633:toxicity*; D010957:Plasmids; D019914:Taq Polymerase
D009944:Organoplatinum Compounds; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D002945:Cisplatin