A DNA binding protein from Xenopus laevis oocyte mitochondria

A DNA binding protein has been isolated, by affinity chromatography on DNA cellulose, from mitochondria and from purified mitDNA-protein complexes from oocytes of Xenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to DNA with a high efficiency. It exhibits an apparently preferential binding to the single-stranded fiber of the D loop structures.     

A DNA binding protein showing sequence specificity for a region containing the replication origin of Xenopus laevis mitochondrial DNA.

In Xenopus laevis mitochondria up to 14 different polypeptides with affinity for the DNA, have been identified by the protein blotting technique. Under stringent binding conditions only one polypeptide displayed specific affinity for a restriction fragment containing the H strand origin of replication of the Xenopus laevis mt chromosome. The proteins were fractionated by double stranded DNA cellulose chromatography. Under conditions which favor high affinity interactions between proteins and DNA, a protein of the 2M NaCl step shows specific binding to the DNA fragments containing the D-loop region. Some physical properties of the protein have been studied. It has a MW of 21.5 Kd and a globular shape as can be inferred from the relationship between MW and sedimentation coefficient (2.7 S). It binds non cooperatively to DNA and forms relatively stable complexes as demonstrated by DNA competition experiments.     

Primary structure from amino acid and cDNA sequences of two Cu,Zn superoxide dismutase variants from Xenopus laevis

A mixture of two different amino acid sequences was discovered in Cu,Zn superoxide dismutase purified from the amphibian Xenopus laevis. No N-terminal post-translational modification was found. The high number of substitutions in the sequence suggested that protein heterogeneity was a product of gene duplication. This was confirmed by isolation of two different cDNA clones. Nucleotide sequence analysis allowed the primary structure of the two peptide chains to be unambiguously assigned. The observed changes (19 in 150 residues) are distributed along the peptide chain to give similar protein net charges although substitutions of the same polarity and/or charge were the exception rather than the rule. The degree of diversity between the two Xenopus variants is comparable to that between mammalian sequences and shows that the putative increase of the rate of mutation for Cu,Zn superoxide dismutase at later evolution stages (Y. M. Lee et al., 1985, Arch. Biochem. Biophys. 241, 577-589; G. J. Steffens et al., 1986, Biol. Chem. Hoppe-Seyler 367, 1017-1024) is observed in amphibians. This is the first time complete sequences for Cu,Zn superoxide dismutase variants from the same organism have been found to be products of divergent genes and not simply allelic mutations.     

Characterization of a Xenopus laevis mitochondrial protein with a high affinity for supercoiled DNA.

A DNA binding protein of 31 Kd -mtDBPC- has been isolated from X. laevis oocyte mitochondria. It is present in large amounts in the organelle and does not show any enzymatic activity. Its binding to the superhelical form of a DNA is higher than for any other form, or for RNA. No sequence specificity could be found for any mtDNA fragments tested, including both origins of replication. It is able to introduce superhelical turns into relaxed circular DNA in the presence of a topoisomerase I activity. It could be a component of the mitochondrial nucleoids.     

A restriction map of Xenopus laevis mitochondrial DNA

The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.     

DNA binding protein from ovaries of the frog, Xenopus laevis which promotes concatenation of linear DNA

A soluble extract of Xenopus laevis ovaries catalyzed ATP-dependent concatenation of linear duplex DNA molecules. DNA ligase and a unique X. laevis DNA binding protein were required for the formation of concatemers. A linear DNA concatenation system was reconstituted using T4 DNA ligase and homogeneous X. laevis DNA binding protein. This system catalyzed intermolecular ligation of DNA molecules into linear concatemers of up to ten or more times monomer length.     

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

The binding of the Xenopus laevis mitochondrial protein mtDBP-C to DNA was studied by equilibrium density banding, agarose gel electrophoresis and electron microscopy. The results obtained show that the mtDBP-C binds cooperatively to DNA irrespective of whether the DNA is supercoiled, relaxed or linear and it induces the formation of superhelical turns locally leading to the formation of a highly folded structure. It appears that this protein could be involved in the compaction of DNA in the mitochondrial nucleoid.     

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Cloning,sequencing and expression of the two genes encoding the mitochondrial single-stranded DNA-binding protein in Xenopus laevis

In Xenopus laevis the single-stranded DNA binding protein imported into the mitochondria consists of two highly related polypeptides. The establishment of the genomic nucleotide sequences reveals that they are encoded by two different genes, XLSSB1 and XLSSB2. The deduced amino acid sequence is identical to the direct amino acid sequence determined by Edman degradation of the mitochondrial polypeptides [Ghrir, R., Lecaer, J.P., Dufresne, C. and Gueride, M. (1991) Primary structure of the two variants of Xenopus laevis mtSSB, a mitochondrial DNA binding protein. Arch. Biochem. Biophys. 291, 395–400]. Both genes are organized in seven exons and six introns, the sequence of the peptide leader is interrupted by an intervening sequence (intron 2). The exon/intron junctions are in exactly conserved positions, splitting the same codon. A high level of identity is observed between corresponding introns of the two genes over part or most of their lengths. Structural features of intronic sequences reveal multiple rearrangements and exchanges during the evolution of X. laevis species. A CCAAT box and the potential regulatory elements NRF-2 and Sp 1 are observed in the 5′-flanking region of both genes. During oogenesis, XLSSB gene expression is correlated with the replicative activity of the mitochondrial DNA.     

Circular mitochondrial DNA from Xenopus laevis and Rana pipiens

Mitochondrial DNA from oocytes of Xenopus laevis and Rana pipiens and from the liver of Gallus domesticus was studied by electron microscopy using the Kleinschmidt technique. A high percentage of circular molecules, either highly twisted or open, was observed in all preparations. The mean contour length of circles from X. laevis was 5.40 , from R. pipiens 5.56 and from G. domesticus 5.26 . Highly twisted circles were found in greater abundance in a fresh preparation than in preparations left standing for 3 months. These molecules are considered to be the native form of mitochondrial DNA.     

Effects of the Xenopus laevis mitochondrial single-stranded DNA-binding protein on the activity of DNA polymerase gamma

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 gamma. Its properties suggest that it is involved in strand displacement during the replication of the mitochondrial genome.     

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.     

Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.     

Modelling the three-dimensional structure and electrostatic potential field of the two Cu,Zn superoxide dismutase variants from Xenopus laevis

The crystallographic structure of bovine superoxide dismutase has been used as a template for the graphic reconstruction of the three-dimensional structures of the two Xenopus laevis variants (Schininà, M.E. et al. Arch. Biochem. Biophys. 272:507-515, 1989). In these models the structure-essential residues maintain their position and their structural role, and the interactions between the subunits and the close packing within the beta-barrel are maintained with conservative substitutions and even increased with "aromatic pairs." Because of the same topological motif and surface location of charges, arising from the model building of the two variants with respect to the bovine enzyme, we have calculated the electrostatic potential fields around the models of the two Xenopus laevis variants by numerically solving the Poisson-Boltzmann equation. We show that conservation of a specific space-relationship of charges maintains the potential field pattern already observed in the bovine enzyme, where a negative potential field surrounds the protein surface and specific positive regions wrap up the copper center active site. This electrostatic potential field distribution supports the idea that electrostatic interactions control, like in the bovine enzyme, the mechanism of enzyme-substrate recognition in the Xenopus laevis Cu,Zn superoxide dismutases, suggesting that coordinated mutation of charged residues has occurred in the evolution of this enzyme.     

Repair of DNA damage in a mitochondrial lysate of Xenopus laevis oocytes.

We examined DNA repair activities of a mitochondrial lysate derived from Xenopus laevis oocytes. Plasmid DNA, exposed to HCl, H2O2 or UV light, was used as the substrate for the in vitro repair reaction. DNA synthesis in the lysate was stimulated 2-8-fold by such lesions, indicating the presence of excision repair activities. This repair DNA synthesis was not affected by aphidicolin, but was sensitive to N-ethylmaleimide. Thus the mitochondrial DNA polymerase, i.e., pol gamma is indeed involved in the reaction. Actual repair of the depurinated DNA was demonstrated by using the polymerase chain reaction (PCR), where the amount of the amplified DNA fragment increased significantly if the depurinated template was incubated in the lysate prior to the PCR. UV-irradiated DNA, on the other hand, restored its ability as a PCR template only if the repair reaction was carried out under the light. Therefore, in this system, UV-induced damage is repaired mainly by photoreactivation. These results show that mitochondria of Xenopus oocytes possess excision repair as well as photolyase activities, and that the in vitro repair system described here should be useful for further molecular characterization of such DNA repair machinery.     

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB)

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.