"Action-at-a distance" of a new DNA oxidative damage product 6-furfuryl-adenine (kinetin) on template properties of modified DNA.
N(6)-furfuryladenine (kinetin, K) was shown to have cytokinin activity and antiageing effects. It also appears to protect DNA against oxidative damage mediated by the Fenton reaction. Kinetin was identified as a natural component of DNA in plant extract, calf thymus DNA, fresh DNA preparations from human cell culture, as well as in human urine. A proposed mechanism of kinetin synthesis includes furfural, the oxidative damage product of a 2-deoxyribose moiety of DNA, which reacts with an adenine residue to form N(6)-furfuryladenine at DNA level. The identification of kinetin in plant cell extracts, as well as human urine, suggests its excision from DNA by repair mechanisms. Since such a bulky modification as kinetin induces conformational changes of DNA, this could lead to mutations. Therefore, it was interesting to analyze an effect of kinetin on coding properties of DNA. Chemically synthesized oligodeoxynucleotide (20-mer) containing kinetin AAAACTGCCGTCCTGAKGAT was used as a primer. It was elongated in a polymerase chain reaction (PCR) on a template plasmid pEW1 harboring a 210-bp fragment of DNA derived from the 5' end of HIV mRNA. The PCR product of that length containing kinetin in position 17 from the 5' end was isolated and sequenced. Interestingly, DNA polymerase correctly incorporates thymine opposite of kinetin (an adenine derivative) on the complementary strand, but the misincorporations occur in a vicinity of the modified base.
Wyszko
Eliza
E
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznań, Poland.
Barciszewska
Mirosława Z
MZ
Markiewicz
Maria
M
Szymański
Maciej
M
Markiewicz
Wojciech T
WT
Clark
Brian F C
BF
Barciszewski
Jan
J
D000225:Adenine / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D002855:Chromatography, Thin Layer; D004249:DNA Damage; D016325:HIV Long Terminal Repeat; D007701:Kinetin; D009841:Oligonucleotides / Q000138:chemical synthesis; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D009841:Oligonucleotides; D000225:Adenine; D007701:Kinetin