Neocarzinostatin-mediated DNA damage in a model AGT.ACT site: mechanistic studies of thiol-sensitive partitioning of C4' DNA damage products.
Double-strand (DS) DNA damage caused by neocarzinostatin (NCS) has been studied in the trinucleotide AGT-ACT sequence in an AP-1 transcription factor binding site. There are strong similarities between bistranded lesions produced at AGT.ACT and AGC-GCT, including the fact that DS lesions outnumber SS lesions on the AGT and AGC strands, while SS exceed DS on the ACT and GCT strands. Structure-function studies revealed that a variety of different thiols produced bistranded lesions in this model by predominantly C4'-hydrogen atom abstraction (84-93%) at the T of AGT and C5'-hydrogen atom abstraction (87-91%) at the T of ACT. Single-strand (SS) lesions were found to represent a variable mixture of C4' and C5' chemistry. The C4'-hydroxylated abasic site occurred in both SS and DS lesions at both sites and accounted for most of the DS damage at AGT (60-83%); the remaining damage consisted of 3'-phosphoglycolate- and 3'-phosphate-ended fragments. The nature of the thiol was found to affect the partitioning of the breakdown products arising from C4' and, to a lesser extent, C5' hydrogen atom abstraction. Production of 3'-phosphoglycolate residues, restricted mainly to the T of AGT in bistranded lesions, correlated with the incidence of direct DS breaks in the AGT.ACT model and in plasmid DNA and appeared to be influenced by the reducing power of the thiol activator. Furthermore, hydrazine and sodium borohydride both inhibited the formation of glycolate, an effect that was exploited to determine the rate constant for 3'-phosphoglycolate formation: 0.06 min-1 at 0 degree C, pH 7.4. Under anaerobic conditions, the nitroaromatic radiation sensitizer misonidazole caused a large increase in glycolate production in both SS and DS lesions formed by NCS, which suggests that the formation of 3'-phosphoglycolate, like 3'-formylphosphate generated by C5' chemistry, involves an oxyradical intermediate. The pathways for DNA damage involving C4' and C5' hydrogen atom abstraction thus share many common features, several of which are consistent with a mechanism for the production of NCS-mediated bistranded lesions at AGT.ACT that involves a tetraoxide bridge joining the lesions on opposite strands of DNA.
Dedon
P C
PC
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Jiang
Z W
ZW
Goldberg
I H
IH
D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D006016:Glycolates / Q000378:metabolism; D007700:Kinetics; D008920:Misonidazole / Q000494:pharmacology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010957:Plasmids; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000633:toxicity*; D014157:Transcription Factors / Q000378:metabolism; D009353:Zinostatin / Q000378:metabolism / Q000633:toxicity*
D004277:DNA, Single-Stranded; D006016:Glycolates; D009711:Nucleotides; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; D008920:Misonidazole; D009353:Zinostatin