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Maintaining of mitochondrial genome plays an important role in viability of whole organism. Mutations in mtDNA lead to progression of many diseases such as muscular dystrophies and neurodegenerative disturbances. Moreover, some deletions in mtDNA are linked with accelerated aging or death. The main pathway to prevent deletions in mtDNA caused by double-strand DNA breaks is a homologous recombination. The presence of such mechanisms was shown for various organisms, e.g. fungi, protists, invertebrates, and mammals. It is believed that mechanisms of mitochondrial homologous recombination are conservative from yeasts to human, and some of components of this system were identified. Nevertheless, the whole molecular mechanism of homologous recombination in mitochondria is still unclear. As it was previously shown by indirect experiments, important role in yeast mitochondrial recombination belongs to major nucleoid protein, Abf2p. Deletion of its gene leads to increased mtDNA deletion rate caused by intramolecular recombination, and decrease in Holliday’s junction formation. Moreover, Abf2p was shown to participate in mtDNA copy number control, its replication and inheritance. In our study we have determined structural specificity and some physico-chemical parameters of Abf2p binding to DNA. We have shown that Abf2p specifically binds to cruciform DNA, in contrast to unspecific binding to linear double-stranded DNA, 5’- and 3’-overhangs, gaps, nicks, 3’-invasion–like structures, and ss- and ds-forks. By EMSA approach we have determined apparent dissociation constants for Abf2p complexes with listed above structures. These constants are from ~100 nM (for dsDNA) to ~10 nM (for Holliday junction). We have also determined the length of Abf2p binding site on DNA as 13 bp. Finally, by two independent techniques we have shown that Abf2p binds to any DNA structure in monomeric state that differs yeast protein from its human homolog TFAM, which binds DNA as a dimer.