Effects of the Xenopus laevis mitochondrial single‐stranded DNA‐binding protein on the activity of DNA polymerase γ

Abstract

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...

The Xenopus laevis mitochondrial protein mtDBP-C cooperatively folds the DNA in vitro.

“…The mtDBP-C inhibits mitochondrial transcription in vitro probably through local modification of supercoiling (Barat-Gueride et al, unpublished results) which suggests that it could be involved in this process. Since, unlike the protein mtSSB (Mignotte et al, 1988), it does not stimulate the DNA synthesis catalysed by the X. laevis DNA polymerase -y (Mignotte et al, unpublished results), it is probably not involved in the elongation step of the DNA replication process. However, by analogy with the action of the prokaryotic histone-like protein HU (Dixon and Kornberg, 1984) it could participate, through higher order protein-DNA structures, in the initiation of the replication of mtDNA which occurs in highly supercoiled molecules (Callen et al, 1983).…”

Mitochondrial Single-stranded DNA-binding Protein from Drosophila Embryos

“…To date, however, the effects of mtSSB on the function of mitochondrial DNA polymerase are unclear. The rat (9) and frog (10) mtSSBs have been shown to stimulate partially purified forms of pol ␥ on homopolymeric DNA substrates, yet the frog protein was found to be completely inhibitory to mitochondrial DNA polymerase activity on singly primed single-stranded viral DNA (10). More recently, an SSB isolated from human mitochondria was shown to enable highly purified human pol ␥ to utilize singly primed M13 DNA (11).…”

“…Consistent with a role in mtDNA replication, interactions between mtSSB and other mitochondrial replication proteins have been observed. In vitro studies indicate that under some conditions, the rat and X. laevis mtSSBs stimulate partially purified forms of mitochondrial DNA polymerase (9,10), and a putative human mtSSB stimulates human pol ␥ (11). In addition, genetic evidence from yeast suggests an interaction between RIM1 and the mtDNA helicase, PIF1 (7).…”

Mitochondrial Single-stranded DNA-binding Protein Is Required for Mitochondrial DNA Replication and Development inDrosophila melanogaster

“…mtSSB binds to single-stranded DNA (ssDNA), coating the displaced ssDNA, which is the template for lagging DNA strand synthesis in mtDNA replication, and preventing it from renaturation . mtSSB also stimulates greatly the activity of pol ␥ from various species (Mignotte et al, 1988;Hoke et al 1990;Williams and Kaguni, 1994;Thöm-mes et al, 1995). Although the mtSSB gene was shown to be essential in yeast (van Dyck et al 1992), its role in mitochondrial biogenesis in animals has not been explored, despite the fact that it represents a candidate gene for inherited human mtDNA depletion.…”