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The mhg1 (NCU02695/ ada-23) gene encodes the mitochondrial high-mobility group box (HMG-box or HMGB) protein in Neurospora crassa. The mhg1 KO strain (mhg1 KO) has mitochondrial DNA (mtDNA) instability and a short lifespan; however, the function of MHG1 remains unclear. To investigate the role of this protein in the maintenance of mtDNA, domain deleted MHG1 proteins were expressed in the mhg1 KO strain, and their effects were analyzed. We identified two putative HMG-domains, HMGBI and HMGBII. Although deletion of the HMG-box did not abolish MHG1's mitochondrial localization, the mhg1 KO phenotype of a severe growth defect and a high sensitivity to mutagens could not be restored by introduction of HMG-box deleted mhg1 gene into the KO strain. It was indicated that recombinant full-length MHG1, i.e., mitochondrial targeting sequence (MTS) containing protein, did not exhibit explicit DNA binding, whereas the MHG1 protein truncated for the MTS sequence did in vitro by an electrophoretic mobility shift assay. Furthermore, recombinant MHG1 protein lacking MTS and HMG-domains, either HMGBI or HMGBII, had DNA affinity and an altered band shift pattern compared with MTS-truncated MHG1 protein. These results suggest that cleavage of MTS and appropriate DNA binding via HMG-domains are indispensable for maintaining mtDNA in N. crassa. • Two high-mobility group domains are required for the function of MHG1. • The two high-mobility group domains have different DNA-binding properties. • Neither high-mobility group domain recapitulates the DNA-binding properties of MHG1. • Cleavage of the MTS motif is necessary for the interaction between MHG1 and DNA. [ABSTRACT FROM AUTHOR]