Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (I) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.     

Male transmission of linear plasmids and mitochondrial DNA in the fungus Neurospora

One of the general rules of heredity is that in anisogamous matings genetic elements in organelles are inherited maternally. Nevertheless, there are cases of paternal transmission, both as rare exceptions, and as regular modes of inheritance. We report two new cases of paternal transmission in crosses of the model fungus Neurospora. First, we show leakage of a linear plasmid from males, the first case in fungi and the second in eukaryotes. Transmission frequencies ranged from 1% to 15% in different crosses, but some crosses showed no detectable male transmission. Second, we show leakage of male mitochondrial DNA (mtDNA), the second case in fungi. Some of the resulting progeny have only the male mtDNA type, but some are heteroplasmons. Heteroplasmons show novel restriction fragments attributable to recombination or rearrangement. Heteroplasmy of mtDNA through male transmission has not been reported previously in any eukaryote. In addition we have shown paternal leakage of circular mitochondrial plasmids, supporting another reported case. In a male bearing a linear and a circular plasmid, these plasmids and the mtDNA are transmitted in different combinations. These results show a potential for mitochondrial segregation and assortment during the sexual cycle in anisogamous fungi, pointing to more potential avenues for novel associations between genomic compartments, and between genomic and extragenomic elements.     

Muscle creatine kinase-deficient mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function

Functional properties of in situ mitochondria and of mitochondrial creatine kinase were studied in saponin-skinned fibers taken from normal and M-creatine kinase-deficient mice. In control animals, apparent Km values of mitochondrial respiration for ADP in cardiac (ventricular) and slow-twitch (soleus) muscles (137 +/- 16 microM and 209 +/- 10 microM, respectively) were manyfold higher than that in fast-twitch (gastrocnemius) muscle (7.5 +/- 0.5 microM). Creatine substantially decreased the Km values only in cardiac and slow-twitch muscles (73 +/- 11 microM and 131 +/- 21 microM, respectively). As compared to control, in situ mitochondria in transgenic ventricular and slow-twitch muscles showed two times lower Km values for ADP, and the presence of creatine only slightly decreased the Km values. In mutant fast-twitch muscle, a decrease rather than increase in mitochondrial sensitivity to ADP occurred, but creatine still had no effect. Furthermore, in these muscles, relatively low oxidative capacity was considerably elevated. It is suggested that in the mutant mice, impairment of energy transport function in ventricular and slow-twitch muscles is compensated by a facilitation of adenine nucleotide transportation between mitochondria and cellular ATPases; in fast-twitch muscle, mainly energy buffering function is depressed, and that is overcome by an increase in energy-producing potential.     

Role of caspases (ICE/CED 3 proteases) in DNA damage and cell death in response to a mitochondrial inhibitor, antimycin A

Caspases (ICE/ Ced3 proteases) are a closely related family of cysteine proteases that play a key role in apoptotic cell death. We examined the role of caspases in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A. LLC-PK1 cells contain caspase activity that was markedly inhibited by cleavage site-based peptide inhibitors of caspases but not by inhibitors of serine, cysteine, aspartate or metalloproteinases. The caspase activity increased within five minutes of exposure to antimycin A, preceding any evidence of DNA damage and cell death. The specific caspase inhibitors. Ac-Tyr-Val-Ala-Asp-aldehyde (inhibitor I) and Ac-Asp-Glu-Val-Asp-aldehyde (inhibitor II) prevented, in a dose dependent manner, antimycin A-induced DNA strand breaks as determined by DNA unwinding assay (residual double stranded DNA in control, 94 +/- 2%; antimycin A alone, 48 +/- 3%; antimycin A + inhibitor I at 50 microM, 93 +/- 2%; antimycin A + inhibitor II at 50 microM, 89 +/- 5%; N = 3 to 4, P < 0.001). These inhibitors also prevented antimycin A-induced DNA fragmentation as determined by agarose gel electrophoresis and by in situ labeling of cell nuclei by the terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) method. The caspase inhibitors markedly prevented antimycin A-induced cell death in a dose-dependent manner as measured by trypan blue exclusion (control 6 +/- 1%, antimycin A alone 40 +/- 1%, antimycin A + inhibitor I at 50 microM 16 +/- 1%, antimycin A + inhibitor II at 50 microM 16 +/- 1%; N = 4 to 7, P < 0.001). These data indicate that the caspase family of enzymes play an important role in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A.     

Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.     

The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in rho+ x rho0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of rho+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a rho+ x rho+ cross, in which there is little mixing of parental mtDNAs, Abf2p-GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In rho+ x rho0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.     

The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance

In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.     

Ageing-associated large-scale deletions of mitochondrial DNA in human hair follicles

Hair follicles plucked from the bi-temporal region of the scalp of 433 Chinese subjects of different ages were used for the examination of ageing-associated mutations of human mitochondrial DNA (mtDNA). By use of PCR techniques, we detected the 4,977 bp and 7,436 bp deletions of mtDNA in hair follicles from aged individuals. The frequencies of occurrence of both mtDNA deletions were found to increase with age of the subject. Moreover, we employed a semi-quantitative PCR method to determine the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in hair follicles. The results showed that the average proportion of the 4,977 bp dmtDNA in hair follicles were 0.05% +/- 0.01%, 0.00%, 0.55% +/- 0.05%, 0.52% +/- 0.24%, 0.65% +/- 0.17%, 1.33 +/- 0.25%, and 1.89% +/- 0.81% for the subjects in the age groups of 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-99, respectively. Furthermore, we screened all the subjects harboring the 4,977 bp and/or 7,436 bp deletions for tandem duplications in the D-loop region of mtDNA by PCR with back-to-back primers. The results showed that none of the previously reported tandem duplications were present in all the hair follicles examined. This indicates that tandem duplications do not predispose to large-scale deletions of mtDNA. However, the data suggest that mtDNA deletions occur and accumulate in hair follicles during human ageing. As hair follicles can be easily and non-invasively obtained from the human, we suggest that the aged-dependent accumulation of dmtDNAs in hair follicles may be used for the monitoring of human ageing process.     

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.     

Sequence polymorphism of mitochondrial DNA control region in Japanese

Sequence polymorphisms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 100 unrelated Japanese were determined by PCR amplification and direct sequencing. Sequences of 404 nucleotides for hypervariable region I and 379 nucleotides for region II were obtained. Variable sites (85 and 45) were revealed in region I and region II, respectively, as compared to the reference sequence, and a total of 96 different genetic patterns from both regions I and II were determined. A point mutation heteroplasmy was observed at the ratio of approximately 50:50 from one individual at the sequence position 151 showing a nucleotide transition from C to T. The probability of identity was estimated as 2.3% for region I, 3.9% for region II, and 1.1% combined for both regions. These results suggest that sequence polymorphism of mtDNA control region would be very useful in forensic practice as a marker for individual identification.     

Fragmentation of human heart mitochondrial DNA associated with premature aging

Point mutations, oxygen damage and deletions in the heart mitochondrial (mt) DNA of a 19-year-old male patient with premature aging, who died of mitochondrial cardiomyopathy, were comprehensively analyzed. With total base-sequencing, one syn- mutation in the tRNA(Asp) gene and one mit-mutation in the ND3 gene were demonstrated. Using microHPLC/MS, 0.20% of the total deoxyguanosine (dG) were proved to be converted into its hydroxy-radical adduct, 8-hydroxy-dG, of which amount corresponds to that in normal subjects of 78 years old. The total detection system for mtDNA deletions, using 180 kinds of primer pairs, revealed extensive fragmentation of mtDNA; 235 types of deletions existed with various sizes, 97 of which yielded mtDNA minicircles lacking both of the replication origins of light- and heavy-strands. Deleted mtDNA accounted for 84% of the total mtDNA. In a man died from an accident at age 28 having almost the same mtDNA genotype except syn-, 50 types of deleted mtDNA, accounting for 15% of the total, were detected in his heart mtDNA. These results will present a clue to an unidentified mechanism of somatic mtDNA replication and the molecular basis of aging heart.     

In situ characterization of islets in diabetes with a mitochondrial DNA mutation at nucleotide position 3243

Changes in the pancreas of diabetic patients with the A-to-G mitochondrial DNA (mtDNA) mutation at nucleotide position 3243 base pair (bp) have not previously been described. The clinical phenotypes of diabetes associated with the mtDNA 3243 mutation range from NIDDM to IDDM. We sought the presence of the mutation and studied volume of beta-, alpha-, and delta-cells, mitochondrial enzyme activity, and presence of apoptosis in diabetic pancreases obtained at autopsy. Pancreases were obtained from 16 patients with IDDM, from 18 patients with NIDDM, and from 11 nondiabetic patients. Mitochondrial enzyme activity was determined for cytochrome c oxidase (COX), the subunits of which are partially encoded by mtDNA, and for succinate dehydrogenase (SDH), the subunits of which are solely encoded by nuclear DNA. The volumes of islet beta-, alpha-, and delta-cells were estimated by computerized morphometry. Pancreatic cells were examined for apoptosis by an in situ end-labeling procedure. The mtDNA 3243 mutation was detected in 1 of 16 (6%) pancreases from the IDDM patients; none of the pancreases from 18 NIDDM patients and 11 nondiabetic patients had the mutation. The single patient with the mtDNA 3243 mutation was a 56-year-old woman with IDDM, aged 39 years at diabetes onset, whose mother was diagnosed with NIDDM. The patient had a history of secondary failure of oral hypoglycemic agents and had a marked decrease in the number of beta-cells. The islet beta-cells and non-beta-cells of the patient showed extremely decreased COX enzyme activity. The islet cells in the patient showed a high activity when examined for SDH. Some pancreatic exocrine cells also showed decreased COX activity with high SDH activity. In IDDM, NIDDM, and nondiabetic patients without the mtDNA 3243 mutation, only weak staining for SDH of the islet cells showed. The percentage of heteroplasmy of the mtDNA 3243 mutation in pancreatic micropunched islet specimens was 63 +/- 5% (mean +/- SD) in the islets, 32 +/- 3% in the exocrine pancreas, and 8 +/- 1% in peripheral polymorphonuclear cells. Apoptotic cells were not observed in the IDDM pancreas in the patient with the mtDNA 3243 mutation. The fact that higher levels of mutated mtDNA at 3243 bp were found in affected islets rather than in other tissue suggests that the distribution of the mutant may determine the effect on islet function. A characteristic decrease in the mitochondrial enzyme with COX activity and accelerated SDH activity of the affected islets may provide new insights into the pathogenesis of mitochondrial diabetes.     

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.     

In vivo determination of replication origins of human mitochondrial DNA by ligation-mediated polymerase chain reaction

A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5' ends of true replicate from those of abortive ones, we examined the 5' ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5' ends. Treatment of DNA with RNase H frees 5' ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.     

Induction of mammalian DNA topoisomerase I-mediated DNA cleavage and DNA winding by bulgarein

Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist.     

Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency

The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to introduce mtDNA from non-human apes into a human cell line lacking mtDNA (rho degrees), and succeeded in producing human-common chimpanzee, human-pigmy chimpanzee, and human-gorilla xenomitochondrial cybrids (HXC). Here, we present a comprehensive characterization of oxidative phosphorylation function in these cells. Mitochondrial complexes II, III, IV, and V had activities indistinguishable from parental human or non-human primate cells. In contrast, a complex I deficiency was observed in all HXC. Kinetic studies of complex I using decylubiquinone or NADH as limiting substrates showed that the Vmax was decreased in HXC by approximately 40%, and the Km for the NADH was significantly increased (3-fold, p < 0.001). Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone produced a mild effect in control cells (0-10% inhibition) but produced a marked inhibition of HXC respiration (50-75%). Immunoblotting analyses of three subunits of complex I (ND1, 75 and 49 kDa) showed that their relative amounts were not significantly altered in HXC cells. These results establish HXC as cellular models of complex I deficiency in humans and underscore the importance of nuclear and mitochondrial genomes co-evolution in optimizing oxidative phosphorylation function.