Book of Abstracts: Albany 2007
June 19-23 2007
In vitro Effect of Modified TFO on the DNA-gyrase Activity and Binding with Target Duplex
Synthetic pyrimidine oligodeoxynucleotide (TFO) can recognize oligopurine-oligopyrimidine tract of double-stranded DNA in a sequence-specific manner and bind in the major groove as third strand, forming local triple helix. The stability of pyrimidine/parallel motif triplexes, containing two canonical T·A∗T and C·G∗C+ base triplets, is pH-dependent because of the requirement for third strand cytosines? protonation at N3 positions (pKa = 4.6). To overcome such a low pH restriction triplex formation can be facilitated by third strand cytosines? modifications.
The influence of TFO (32-nt) on the binding of DNA-gyrase from S. noursei and cleavage reaction in vitro has been examined. The target duplex represented 162 bp DNA-fragment from pBR322 containing a unique site of DNA-gyrase. We have found that the replacement of TFO-cytidines by C-nucleoside of 2-aminopyridine makes possible to form a stable triplex under the physiological conditions (pH 7-7.5). Triplex formation was supported using melting temperature measurements and gel mobility shift analysis.
To investigate the effect of TFO on gyrase activity the cleavage reaction of the enzyme on the 162-bp fragment was analyzed by gel electrophoresis. The super-coiling reaction, catalysed by DNA-gyrase, required ATP, Mg2+, ions and pH 7-8. The results of the enzymatic experiments in vitro demonstrated, that the cleavage reaction, mediated by DNA-gyrase, can be efficiently inhibited by this modified TFO. It was shown that triple helix formation directly blocks the unique specific cleavage site, but does not prevent the binding of enzyme from its whole binding region. On the other hand, a sequence-specific minor groove binder chromomycin A3 inhibits the cleavage of the 162-bp fragment and displace the enzyme from DNA binding. Thus, both the major and minor grooves play a role in the enzyme-DNA interaction and cleavage reaction.
Supported by grants of BMFT BEO 21/0311109 and DFG 436 RUS 113/40/13 (Germany).
Anna Gabrielian1, 2, *
1Institute of Fine Organic Chemistry of Armenian National Academy of Sciences