The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated.

Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a protease-insensitive, submolecular domain (Mr approximately equal to 6,000) that retained the capacity to bind tenaciously to the DNA. Analysis of chemically fixed complexes by CsCl isopycnic gradient centrifugation showed that P16 was bound to a large subpopulation of mtDNA enriched in displacement loops (D-loops). Based upon the effective buoyant density of the complex in CsCl gradients and the molecular weights of P16 and mtDNA, it was estimated that a mean of 49 P16 molecules were bound per mtDNA. For this measurement, the variation in hydration of protein and DNA at different CsCl concentrations was ignored. Analysis of restriction endonuclease-digested complexes by glass fiber filters that bind only protein-associated DNA resulted in the retention of a single fragment regardless of the enzyme, or enzymes, used. In each case, the retained fragment was the D-loop-containing fragment. With direct electron microscopy, the protein was readily visualized on the displaced single strand portions of D-loops and expanding D-loops. The nucleoprotein fibers were approximately 12 nm in diameter without correcting for the thickness of tungsten coating and roughly 1/3 the length of the double strand segment of the corresponding D-loop structure. In addition, occasional molecules with the characteristics of gapped circles were seen exhibiting a nucleoprotein fibril, presumably containing the single strand gap segment, linking the ends of double strand DNA. P16 was not seen on the double strand portions in any of the complexes.

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.

Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen.

Transient generation of reactive oxygen or nitrogen (ROS/RNS), detected with dihydrodichlorofluoroscein by fluorescence microscopy, occurs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible depolarization of the mitochondrial membrane potential () and decrease in fluorescence of a mitochondria-entrapped dye, calcein, are observed coincidentally. Radiation-induced ROS/RNS, depolarization, and calcein fluorescence decrease are inhibited by the mitochondrial permeability transition inhibitor, cyclosporin A, but not the structural analogue, cyclosporin H. Radiation-stimulated ROS/RNS is also inhibited by overexpressing the Ca(2+)-binding protein, calbindin 28K, or treating cells with an intracellular Ca(2+) chelator. Radiation-induced ROS/RNS is observed in several cell types with the exception of rho(o) cells deficient in mitochondrial electron transport. rho(o) cells show neither radiation-induced ROS/RNS production nor depolarization. We propose that radiation damage in a few mitochondria is transmitted via a reversible, Ca(2+)-dependent mitochondrial permeability transition to adjacent mitochondria with resulting enhanced ROS/RNS generation. Measurements of radiation-induced mitogen-activated protein kinase activity indicate that this sensing/amplification mechanism is necessary for activation of some cytoplasmic signaling pathways by low doses of radiation.

Dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 increases the susceptibility of human leukemia cells (U937) to 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial dysfunction and apoptosis.

The effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction of classic apoptotic morphology, DNA fragmentation, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and underphosphorylation of the retinoblastoma protein (pRb) than their empty-vector counterparts. Enhanced susceptibility of antisense-expressing cells to ara-C was accompanied by a corresponding reduction in clonogenic and suspension culture growth. The increased sensitivity of these cells to ara-C-mediated lethality could not be attributed to cytokinetic perturbations, nor did ara-CTP formation or (ara-C)DNA incorporation differ significantly between the cell lines. Moreover, synchronization of p21 antisense-expressing cells in S-phase by aphidicolin block resulted in a further increase in ara-C-mediated apoptosis, suggesting enhanced drug sensitivity of the S-phase cell fraction. After exposure to ara-C, p21 antisense-expressing cells displayed a greater decline in mitochondrial membrane potential (deltapsi(m)) and generation of reactive oxygen species than their empty-vector counterparts, as well as early potentiation (e.g., within 2-4 h) of cytochrome c release into the cytosolic S-100 fraction. Lastly, ara-C-mediated increases in mitogen-activated protein kinase activity over basal levels were attenuated in p21 antisense-expressing cells. Collectively, these findings indicate that dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 increases the susceptibility of U937 human leukemia cells to ara-C-related lethality, and this phenomenon occurs as a relatively early event that is independent of cell cycle or pharmacodynamic factors and is associated with mitochondrial perturbations implicated in activation of the apoptotic protease cascade.

Expression of mouse Amy-2a alpha-amylase genes is regulated by strong pancreas-specific promoters.

Three types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome. DNA sequence analysis reveals that both classes of Amy-2a genes specify the same unique pancreatic alpha-amylase mRNA species, since they share common exon sequences. Four independently cloned Amy-2a II isolates were found to be identical in all regions sequenced. This suggests that most, if not all, chromosomal Amy-2a II copies are identical. Amy-X is presumably a pseudogene, since its exon sequences, which are distinct from those of Amy-2a, are not detected in pancreatic alpha-amylase mRNA. We have determined the transcriptional activities of the Amy-2a genes by mapping in vitro elongated nascent transcripts to Amy-2a restriction fragments. Transcription initiation occurs at or close to the cap site. The expression of Amy-2a in vivo is under control of strong promoters, which are active exclusively in the pancreas. The accumulation of alpha-amylase mRNA in cells of the exocrine pancreas is regulated mainly at the transcriptional level. We have searched for pancreatic transcripts of Amy-1a, which specifies both parotid gland and liver-type alpha-amylase mRNAs. Surprisingly, the weak Amy-1a promoter, which directs the synthesis of the mRNA containing the liver-type leader sequence, also is active in the pancreas and, hence, in all alpha-amylase-producing tissues.

The gene and processed pseudogenes of the rat mitochondrial single-strand DNA-binding protein: structure and promoter strength analyses.

The gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping genomic clones. The clones encompassed the 5' untranslated sequence and all intron/exon junctions. The gene contained seven exons and six introns. The first exon contained only 5' untranslated sequence. The 16-amino acid mitochondrial targeting presequence, encoded by the second and third exons, was precisely bisected by intron 2. All intron donor and acceptor sites were consistent with the GT/AG consensus. The transcription start site was determined by primer-extension analysis to be 69bp upstream of the translation start codon. The upstream sequence lacked TATA and CCAAT boxes at the expected locations, but did contain several other potential regulatory elements including a GC box (Sp1-binding site) and three NRF-2 sites, one of which was located precisely beside the transcription start site. A 10 out of 12 imperfect NRF-1 site was located within the first exon. The 5' flanking sequence (-546 to +30) was shown to have strong promoter activity in transient transfection assays in primary rat hepatocytes and HepG2 cells. In addition, evidence for the existence of several mtSSB processed pseudogenes was obtained. These pseudogenes lacked introns and contained substitution and deletion mutations compared to the cDNA sequence. The 5' upstream region of one of the pseudogenes was analyzed and found to contain negligible promoter activity.

Rearrangements in the shorter arc of rat mitochondrial DNA involving the region of the heavy and light strand promoters.

Brain mtDNA from rats ranging in age from 1 day to 33 months were analyzed for large-scale rearrangements using nested PCR. The region of the mtDNA targeted by the primers was the shorter are between the two origins of replication and encompassed the heavy (H) and light (L) strand promoters (HSP) and (LSP). Rearrangements lacking 4 to 5 kb of genomic sequence were found in animals of all ages. Twenty-two different rearrangements were sequenced; two of these were found replicated in several different animals. All the rearrangements identified lacked an HSP and six lacked an LSP as well. The end points of each rearrangement had short direct repeats of 9 bp or less, but repeats of 4 bp or less were the most common. The mode of involvement of the direct repeats in the rearrangement mechanism varied since in some cases a sequence precisely equivalent to one member of the paired repeats was found at the junction; whereas in other cases, more or less than one complete member was found. Sixteen of the 22 rearrangements terminated on one side within a 22-bp locus, or hot spot, located at a potential stem-loop structure midway between the HSP and LSP. The other ends of these rearrangements were at different sites. In addition, a secondary hot spot was found near the junction between the tRNA(Ala) and tRNA(Asn) genes, which lie in a cluster of five tRNA genes that surround the stem-loop structure of the L-strand origin of replication. The data suggest a link between secondary structure and short direct repeats and the rearrangement mechanism(s). The results of this study, in conjunction with out previous study of the longer arc of rat mtDNA (Van Tuyle, G.C., J.P. Gudikote, V.H. Hurt, B.B. Miller and C.A. Moore (1996) Multiple, Large deletions in rat mitochondrial DNA: Evidence for a major hot spot, Mutation Res., 349, 95-107), indicate that nearly the entire mitochondrial genome is subject to rearrangement mutations that are detectable in brain tissue throughout an animal's life span.

Mitochondrial DNA rearrangements, including partial duplications, occur in young and old rat tissues.

Using polymerase chain reaction (PCR) with back-to-back primers, 85 different mitochondrial DNA (mtDNA) rearrangements, consisting of partial duplications or mini-circles, were detected in brain, liver, and heart tissue from Fischer 344 rats. The regions around the mitochondrial tRNALeu(UUR) gene, the cluster of three tRNA genes [His, Ser(AGY), Leu(UUC)], as well as the region of the displacement loop were analyzed separately with different primer sets. Rearrangements were detected in all regions analyzed in samples taken throughout the animal life span, ranging from 1 day old to 33 months of age (senescent). Two-thirds of the rearrangements terminated at short (3-9-bp) direct repeats. Three of the different rearrangements were detected in more than one animal; the most common rearrangement was found in nine different template preparations. Two loci (hot spots) were found to be particularly susceptible to rearrangement, and both were located at sequences that exhibited highly conserved potential for secondary structure formation. The displacement loop region of 10 samples exhibited the presence of multiple tandem duplications ranging between 324 and 449 bp in length. One of these consisted of heterologous, but overlapping, repeating units. Identical PCR protocols were carried out in control experiments using a cloned fragment of mtDNA that encompassed the most common hot spot sequence. The results showed that this fragment did not artifactually generate a rearrangement junction under our PCR conditions and suggested that this sequence does not promote rearrangement mutations in bacteria during the cloning process.

Multiple, large deletions in rat mitochondrial DNA: evidence for a major hot spot.

This study identified 33 different deletions in mitochondrial DNA from four aging Fischer-344 rat brains and from a cultured rat lymphoma cell line (Nb2 cells). The deletions were located in the longer arc between the heavy and light strand origins of replication. PCR products that spanned across the deleted regions were sequenced, and deletions ranging between 6548 bp and 9977 bp in length were identified. Short direct repeats of < or = 8 bp were present at the end points of all but one of the deletions. The remaining deletion contained, instead, a near-perfect direct repeat (9/10 bp) within two base pairs of its end points. In 24 of the deletions, a sequence equivalent to one member of the paired direct repeats was lost with the deleted segment. In the remaining nine, either more or less of the base pairs of a single repeat were lost. Twelve of the 33 different deletions terminated on one side at a common locus (major hot spot) of 5 bp in length, located at the 5' end of the tRNAThr gene. The opposite ends of these 12 deletions were at different sites. The hot spot was located in a region of the mtDNA with strong potential for secondary structure and was flanked by a pair of AT-rich sequences. The utilization of the hot spot as an end point for deletions appeared to be widespread in that it was represented in 1/3-1/2 of the deletions characterized in each of the five mtDNA sources examined. In addition, several minor hot spots, where one end of two or three different deletions coincided, were also identified.

Separation and characterization of 5'- and 3'-tRNA processing nucleases from rat liver mitochondria.

The 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.5 M KCl. Both enzymes were greater than 500-fold purified over the high speed supernatant of a mitoplast extract. The 159-base pre-tRNATyr used as a substrate in this study was synthesized in vitro and contained the Escherichia coli suppressor III tRNATyr plus a 49-base leader sequence and a 25-base trailing sequence. The 5' processing nuclease converted the pre-tRNATyr into two discrete RNA species, identified as the 5'-processed intermediate and the 5' flanking fragment, by endonucleolytic cleavage at the 5' end of the mature tRNATyr sequence. The 3' processing nuclease was inactive with the intact pre-tRNATyr as substrate but efficiently converted the 5'-processed intermediate to the mature tRNATyr, indicating an obligatory order of processing in which 5' maturation was necessary before cleavage by the 3' processing nuclease could occur. The mitochondrial enzymes exhibited optimal activity in the presence of about 2 mM Mg2+, but both enzymes were nearly fully active without addition of exogenous Mg2+ to the reaction mixtures. In contrast, a partially purified 5' processing endonuclease present in the postmitochondrial cytosolic fraction required higher [Mg2+] for activity, thus providing a means for differentiating between these similar enzyme activities obtained from the cytosolic and mitochondrial fractions.

Characterization of a rat liver mitochondrial DNA-protein complex. Replicative intermediates are protected against branch migrational loss.

A stable DNA-protein complex was released from rat liver mitochondria by sodium dodecyl sulfate-lysis and isolated by sedimentation velocity in sucrose density gradients. The mtDNA-protein complex was washed with 0.5 M NaCl and any unbound contaminants were removed by hydroxyapatite column chromatography. The only detectable polypeptide in the complex was a single low molecular weight species (Mr = 16,000) having a slightly basic isoelectric point of 7.6-7.8. Complete digestion of the mtDNA-protein complex with restriction endonuclease HindIII revealed in agarose gels an "extra" band consisting of a subset of the largest fragment population. The fragments in this subset were shown to contain the replicative intermediates which were retarded in electrophoretic migration due to the parental strand separation in the region of the replication loops. No loss of nascent strands due to branch migration of the parental strands was observed upon HindIII cleavage of the covalently closed circular DNA in the mtDNA-protein complex. However, HindIII digestion of completely deproteinized mtDNA resulted in quantitative loss of nascent strands from replicating molecules. These results are interpreted as evidence that the single low molecular weight polypeptide present in the complex plays a major role in maintaining the integrity of replication loops during parental strand scission.

The processing of wild type and mutant forms of rat nuclear pre-tRNA(Lys) by the homologous RNase P.

The 5' processing of rat pre-tRNA(Lys) and a series of mutant derivatives by rat cytosolic RNase P was examined. In standard, non-kinetic assays, mutant precursors synthesized in vitro with 5' leader sequences of 10, 17, 24, 25, and 46 nucleotides were processed to approximately equal levels and yielded precisely cleaved 5' processed intermediates with the normal 7-base pair aminoacyl stems. The construct containing the tRNA(Lys) with the 46-nucleotide leader was modified by PCR to give a series of pre-tRNA(Lys) mutants designed to measure the effect on processing by (1) substituting the nucleotide at the +1 position, (2) pairing and unpairing the +1 and +72 bases, (3) elongating the aminoacyl stem, and (4) disrupting the helix of the aminoacyl stem. Comparative kinetic analyses revealed that changing the wild type +1G to A, C, or U was well tolerated by the RNase P provided that compensatory changes at +72 created a base pair or a G.U noncanonical pair. Mutants with elongated aminoacyl stems that were produced either by inserting an additional base pair at +3:a + 69:a or by pairing the -1A with a +73U, were processed to yield 7-base pair aminoacyl stems, but with different efficiencies. The efficiency seen with the double insertion mutant was higher than even the wild type precursor, but the -1A-U + 73 mutant was a relatively poor substrate. Disrupting the aminoacyl stem helix by introducing a +7G G + 66 mispairing or by inserting a single G at the +3:a position dramatically reduced the processing efficiency, although the position of cleavage occurred precisely at the wild type cleavage site. However, the single insertion of a C at the +69:a position resulted in an efficiently cleaved precursor, but permitted a minor, secondary cleavage within the leader between the -6 and -5 nucleotides in addition to the dominant wild type scission.

Characterization of a DNA primase from rat liver mitochondria.

A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.

Characterization of ribonuclease P isolated from rat liver cytosol.

Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.

Inhibition of mitochondrial DNA synthesis by aflatoxin B1 and dimethylnitrosamine.

The acute and subchronic effects of aflatoxin B1 and dimethylnitrosamine (DMN) on in vitro incorporation of thymidine-3H into DNA (mt-DNA) from mouse liver mitochondria were studied after single injection or after one month or three months of weekly injection. Both carcinogens induced 50% decreases in mt-DNA synthesis acutely, with a 50% decrease in synthesis of high molecular weight mt-DNA. Subchronic administration of aflatoxin B1 inhibited mt-DNA synthesis by 54% with elution profiles similar to the controls. DMN showed only a 29% inhibition of mt-DNA synthesis at one month increasing to 55% at three months.

A compact form of rat liver mitochondrial DNA stabilized by bound proteins.

A highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation coefficient of a highly purified mitochondrial DNA supercoil, and appeared as a stable, heterogeneous population of species ranging in s values between 42 S and about 70 S. Under formamide-spreading conditions most of the rapidly sedimenting forms appeared in the electron microscope as single genome length rosettes constrained at the center in a dense core. Except for an occasional D-loop, no extraordinary structural features were evident along the smooth loops projecting radially from the central core. In sucrose gradients containing various amounts of ethidium bromide, the sedimentation velocity of the folded DNA changed in a biphasic fashion in response to increasing amounts of dye. At a dye concentration of 0.5 microgram per ml the DNA species present reached s value minima, but two major peaks sedimenting at 32 S and 42 S were present at this point. Thus, although these species were similar in superhelix density, there appeared to be additional constraints superimposed upon their tertiary structure that folded these forms to differing degrees of compactness. Direct chemical analyses showed that proteins were bound to the folded DNA at a protein to DNA ratio of about 0.3. Separation of the bound proteins on SDS-polyacrylamide gels revealed an array of proteins ranging in molecular weight between 11,000 and 150,000. Several of the lower molecular weight proteins co-migrated with proteins from the inner mitochondrial membrane, but the major DNA-bound band (Mr = 58,000) was undetectable among the proteins from any other submitchondrial fraction. Digestion of the compact DNA structure with proteinase K under various conditions indicated that the DNA was maintained in the compact conformation by the tightly bound proteins and that the portions of these proteins directly involved in stabilizing the folded DNA were proteinase insensitive unless digestion was carried out in the presence of a disulfide reductant at elevated temperatures.

Benzo[a]pyrene differentially alters mitochondrial and nuclear DNA synthesis in primary hepatocyte cultures.

The incubation of non-proliferating primary cultures of rat hepatocytes with benzo[a]Pyrene for 24 or 48 hours produced a concentration-dependent stimulation of nuclear DNA synthesis (measured as [3H]thymidine incorporation into isolated, purified DNA) whereas the normal processes of D-loop formation and expansion synthesis on mitochondrial DNA were inhibited by as much as 62% of that in control cells. Incubation of cultures with [3H]benzo[a]pyrene showed that highly purified mitochondrial DNA retained 4-15 times more covalently bound adducts per unit length of DNA than did nuclear DNA. We suggest, therefore, that this carcinogenic, environmental pollutant may exert its most profound effects on the mitochondrial, rather than the nuclear, genetic apparatus.

Structural and functional studies of the rat mitochondrial single strand DNA binding protein P16.

The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found. Measurement of the affinity of P16 for isolated sites by titration with either oligo(dT)8 or 5'-dephosphorylated oligo(dT)8 indicated values on the order of 10(7) M-1. P16 exhibited a binding preference for single strand DNA, poly(dT), and poly(dC) in comparison to double strand DNA, poly(U), or poly[d(A-T)]. Although it was not possible to show that P16 destabilizes double helical DNA or even poly[d(A-T)], binding of P16 does inhibit the process of renaturation as shown by inhibition of duplex formation between poly(dA) and poly(dT). The binding of saturating amounts of P16 to single strand poly(dT).oligo(dA)50 template-primers enhanced approximately 10-fold the activity of both the homologous mitochondrial DNA polymerase and the Escherichia coli DNA polymerase I Klenow fragment. However, the mitochondrial DNA primase was nearly completely inhibited by the saturation of the poly(dT) template with P16. Amino-terminal sequence analysis of P16 and a protease-insensitive, DNA binding domain (Mr approximately 6000) revealed that the DNA binding domain residues, at least in part, in the amino-terminal third of the P16 molecule. Furthermore, the amino-terminal sequence was found to be strikingly similar to that of the Xenopus laevis mtSSB-1 and to a lesser extent similar to E. coli SSB and E. coli F sex factor SSB.

Biosynthesis of mitochondrial DNA. Is 8 S DNA an artifact?

Sucrose density gradient fractionation of isolated rat liver mitochondrial DNA ordinarily yields two peaks, one at 39 S, the other at 27 S. However, when these mitochondria are first incubated with a labeled DNA precursor, a labeled peak at about 8 S is also observed. Is this low molecular weight 8 S DNA merely an artifact of contamination or breakdown, or is it a functioning part of the mitochondrial genome? That it is not a nuclear contaminant is shown by: (a) the absence of nuclei or nuclear fragments in active mitochondrial preparations; (b) the insensitivity of 8 S DNA synthesis to treatment of mitochondria with DNase and RNase; (c) the ability of inner membrane preparations to synthesize this DNA; (d) the ability of atractyloside to inhibit incorporation of [3H]dATP into 8 S and 39 S or 27 S DNA equally; (e) the labeling of 8 S DNA (as well as 39 S and 27 S DNA) but not of nuclear DNA after the administration in vivo of [3H]thymidine. The evidence that 8 S DNA is not an artifact resulting from DNA breakdown during mitochondrial incubation or DNA isolation is as follows: (a) 8 S DNA can be isolated from unincubated mitochondrial; (b) 8 S DNA becomes labeled when labeled DNA precursors are administered in vivo; (c) 8 S DNA biosynthesis continues in the complete absence of labeled 39 S or 27 S DNA (whose synthesis is repressed by ethidium bromide), making it unlikely that 8 S DNA is formed from the breakdown of 39 S or 27 S DNA; (d) substitution of milder methods of DNA extraction does not decrease 8 S DNA labeling; moreover, the usual conditions of extraction, when applied to purified 39 S and 27 S DNA, do not generate 8 S DNA, nor does an additional mitochondrial washing cycle; (e) the specific radioactivity of 8 S DNA is higher than that of 39 S or 27 S DNA, making it improbable that the latter forms are precursors of 8 S DNA. Since 8 S DNA is double-stranded, it is not identical to the 7 S fragment of D loop DNA. The hypothesis that the artifactual nicking of those DNA molecules which contain opposing D loops leads to the release of double-stranded fragments was tested. The DNA which was released was predominantly (and probably completely) single-stranded. We conclude that 8 S DNA is probably not an artifact and studies are in progress on its function.