Invasion of Complementary Oligonucleotides into (CA/TG)_(31) Repetitive Region of Linear and Circular DNA Duplexes

摘要

(CA/TG )_n repeats belong to microsatellite DNA. They are the most abundant among the other dinucleotide repeats in mammals, constituting approximately 0.25% of the entire genome. These repeats are recombination hot spots; however, the corresponding mechanisms are yet vague. We postulated that one of the reasons underlying an increase in the recombination frequency in the repetitive region could be the conformational characteristics of duplex resulting from a specific geometry of base-stacking contacts, providing for initiation of a single-stranded DNA invasion in the duplex homologous regions. This work for the first time demonstrates a DNA-DNA interaction of the d(CA)_(10) and d(TG)_(10) oligonucleotides with linear and circular duplexes containing (CA/TG)_(31) repeats during their coincubation in a protein-free water solution at 37°C. Using radioactively labeled oligonucleotides, we demonstrated that the duplex-oligonucleotide interaction intensity depended on the molar ratio of duplex-to-oligonucleotide at a duplex concentration of 30 nM. A decrease in this concentration to 3 nM had no effect on the intensity of oligonucleotide invasion. It was demonstrated that over 1 % of the duplexes yet much less than 10% were involved in the interaction with oligonucleotides assuming that one oligonucleotide molecule interacted with one molecule of the duplex. Analysis of the kinetics showed that d(CA)_(10) invasion commenced from the first minute of incubation with duplexes, while d(TG)_(10) interacted with the duplex even at a higher rate. The role of conformational plasticity of CA/TG repeats in the discovered interaction is discussed as well as its biological significance, in particular, the role of CA microsatellites in the initiation of homologous recombination.