Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the fulllength polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following.

The single-stranded DNA-binding protein from Xenopus laevis oocyte mitochondria, which has been found associated with the D loop, binds to ssDNA in stoichiometric amounts and can under certain conditions stimulate the activity of the DNA polymerase y. Its properties suggest that it is involved in strand displacement during the replication of the mitochondrial genome.The replication of the vertebrate mitochondrial genome is initiated in the only long non-coding region of mtDNA. It proceeds asymmetrically by displacement of the parental H strand (seen as a D-loop structure in the electron microscope) and synthesis of a new H strand hydrogen-bonded to the L strand [l]. A peculiar property of this replication system is that the elongation of the newly synthesized H strand can be stopped at precise sites yielding D loops of various but definite lengths with frequencies usually higher than expected for classical replicative forms (30 -70% in Xenopus hevis oocytes [2]). If the elongation goes through the putative stop signals [3], when two-thirds of the parental H strand are displaced, the origin of light-strand replication becomes exposed and the synthesis of L strand begins in a direction opposite to that of H-strand replication until the formation of two full duplex daughter molecules [l].Now that all the open reading frames of the vertebrate mitochondrial genome have been assigned [4], it is clear that the proteins responsible for the replication of the mtDNA are nuclearly encoded. Various enzymes that are probably involved in this process have been identified [5-121 (and references therein). Among the proteins without any known enzymatic activity that can interact with mtDNA, proteins binding preferentially to the region of the origin of replication have been found [13, 141. Two other DNA-binding proteins, found in X . luevis mitochondria, habe been previously purified and partially characterized. The first protein, called mtDBP C, binds preferentially supercoiled molecules. It could be that it is a structural component of the mitochondrial nucleotids [15]. The other protein, called mtSSB, is found associated with the displaced single strand of D loops [16]. It binds preferentially and cooperatively to single-stranded DNA. Its affinity is higher for pyrimidine-rich sequences and its amino acid composition is close to that of other single-stranded nucleic-acid-binding proteins 1171. This protein is probably homologous to the P16 protein found in rat-liver mitochondria i181.The work presented here has been carried out to characterize further the mtSSB-ssDNA complexes and to specify the possible functions of the mtSSB during mtDNA replication. MATERIALS AND METHODS Muter iulsOvaries were taken from adult female X . luevis bred by Dr D. Boujard (UniversitC de Rennes I).The X . luevis mtSSB was purified as described in [17] except that the phosphocellulose step was omitted and the DNA-cellulose column was replaced by a ssDNA-Ultrogel (I.B.F.) column. This column was eluted stepwise with 0.8 M and 2.5 M NaCl. The mtSSB...