Invasion of complementary oligonucleotides into (CA/TG)31 repetitive region of linear and circular DNA duplexes

摘要

(CA/TG)^sub 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 con-formational characteristics of duplex resulting from a specific geometry of base-stacking contacts, providing for initiation of a single-stranded DNA invasion in th e duplex homologous regions. This work for the first time demonstrates a DNA-DNA interaction of the d(CA)^sub 10^ and d(TG)^sub 10^ oligonucleotides with linear and circular duplexes containing (CA/TG)^sub 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)^sub 10^ invasion commenced from the first minute of incubation with duplexes, while d(TG)^sub 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.[PUBLICATION ABSTRACT]