Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA

Large-scale deletions of mitochondrial DNA (mtDNA) have been described in patients with progressive external ophthalmoplegia (PEO) and ragged red fibers. We have determined the exact deletion breakpoint in 28 cases with PEO, including 12 patients already shown to harbor an identical deletion; the other patients had 16 different deletions. The deletions fell into two classes. In Class I (9 deletions; 71% of the patients), the deletion was flanked by perfect direct repeats, located (in normal mtDNA) at the edges of the deletion. In Class II (8 deletions; 29% of patients), the deletions were not flanked by any obviously unique repeat element, or they were flanked by repeat elements which were located imprecisely relative to the breakpoints. Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II. It is not known how these deletions originate, but both slipped mispairing and legitimate recombination could be mechanisms playing a major role in the generation of the large mtDNA deletions found in PEO.     

Random mtDNA deletions and functional consequence in aged human skeletal muscle

Mitochondrial respiratory chain deteriorates with age, mostly in tissues with high energy requirements. Damage to mitochondrial DNA (mtDNA) by reactive oxygen species is thought to contribute primarily to this impairment. However, the overall extent of random mtDNA mutations has still not been evaluated. We carried out molecular and biochemical analyses in muscle biopsies from healthy young and aged subjects. Deleted mtDNA accumulation was followed by both quantitative PCR analysis to quantify total mtDNA, and Southern-blotting, to determine deleted to full length mtDNA ratio. Enzymatic activities of the mitochondrial respiratory chain were measured in all subjects. Randomly deleted mtDNA appeared mainly in the oldest subjects (beyond 80 years old), affecting up to 70% of mtDNA molecules. The activities of complexes III and IV of the respiratory chain, complexes with mtDNA encoded subunits, are lower in the aged subjects. Physical activity could be one major parameter modulating the mitochondrial respiratory chain activity in aged muscle.     

Statistical evidence for the correlation of DNA deletions in prokaryotic genomes with direct repeats

In the present work a computer analysis of deletion localization in the sequence of the E. coli lacI gene has been carried out by the statistical weight method. Reliable statistical correlation of the deletions location sites with the arrangement of the most perfect direct repeats revealing the shortest distance between repeated fragments has been shown. At the same time statistical analysis did not reveal reliable connection of deletions localization regions with the expected sites of gyrase recognition, sites and other recombination sites. A conclusion has been drawn, that the mechanism of deletions emergence on the basis of repeats appears to be predominant.     

Detection of deletions flanked by short direct repeats in mitochondrial DNA of aging Drosophila

Cumulative damage due to reactive oxygen species (ROS) in mitochondria, especially in mitochondrial DNA (mtDNA), would result in a decrease in mitochondrial respiratory function and contributes to the age-related decline in the physiological functioning of organisms. Previously, we reported the tissue-specific accumulation of deleted mtDNA with age in Drosophila melanogaster. In the present study, to understand the mechanism by which mtDNA deletion is generated with age, nucleotide sequences of deleted mtDNA were determined. Consequently, 33 different sequences each containing a deletion were obtained from flies that were more than 55-day-old. Most of the deletions were found to be flanked by short direct repeats. The present results, together with those from other animals, suggest that there is a common mechanism generating mtDNA deletions through direct repeats.     

Theoretical analysis of mechanisms of occurrence of DNA deletions in prokaryotic genomes based on direct repeats

In the present work a mechanism of deletions emergence on the basis of complementary DNA regions mispairing of direct repeats has been investigated theoretically. A quantitative dependence of the rates of deletions emergence on such parameters of the flanking repeats as the nucleotide composition of repeats, the number of homology damages and the distance between repeated regions has been constructed. It has been proved, that using this relationship one can reliably evaluate the total rates of deletions emergence in the lacI gene sequence of E. coli according to the repeats arrangement in this gene.     

Germ-line deletions of mtDNA in mitochondrial myopathy.

mtDNA encodes subunits of the electron transport chain and is exclusively maternally inherited in mammals. It has been suggested that mtDNA might be the site of some of the mutations causing a group of human disorders called the "mitochondrial myopathies," because these may both be (1) accompanied by defects in the electron transport chain and (2) display a maternal pattern of inheritance. However, all of the deletions and duplications of mtDNA which occur in these patients have been sporadic, apart from families in whom affected members all carry different deletions suggesting a mutant autosomal dominantly inherited nuclear gene with de novo deletions in each individual. We present the first evidence for the presence of deleted mtDNAs in the germ line in these disorders. The patient carries a higher level of deleted mtDNAs than do his relatives, corresponding to severity of symptoms and consistent with a predicted dosage effect. "Selfishness" of deleted mtDNAs is probably one of the factors over and above random segregation of a small number of "founder" mtDNAs (the bottleneck hypothesis) which may be invoked to explain the usual distribution of mtDNAs in different tissues of patients with mtDNA deletions.     

Multiple deletions in mitochondrial DNA at direct repeats of non-D-loop regions in cases of familial mitochondrial myopathy

Muscle mitochondrial DNAs from two brothers with mitochondrial myopathy associated with peripheral neuropathy had multiple deletions, most of which started in non-D-loop regions, unlike in an autosomal dominant mitochondrial myopathy (Zeviani, M. et al., Nature 339, 309 (1989)). The non-D-loop regions with deletions were amplified by the polymerase chain reaction and the resulting fragments were subcloned and then sequenced. At least 12 deletions of different lengths in different sites were found. However, all the deletions were flanked by short direct repeats (4-12 base pairs).     

Two distinct models account for short and long deletions within sequence repeats in Escherichia coli.

In Escherichia coli, (GpC)n sequences cloned into plasmid DNA molecules are deletion-prone with the occurrence of both short (<2 bp) and long (>2 bp) deletion events. These repetitive tracts can be stabilized by interrupting the strict monotony of the repetition with a variant dinucleotide sequence. The stabilization of short deletion events that is mediated by the variant sequence is completely lost in E. coli mismatch repair-deficient strains. In contrast, this repair pathway has no influence on the frequency of occurrence of long deletion events, even in sequences containing the variant repeat. These results lead us to propose two distinct models to account for short and long deletions within repetitive sequences in E. coli. Furthermore, this study reveals that the deletions occur preferentially at the end of the repeat sequence that is distal with respect to the origin of replication.     

Two ubiquitin-long-tail fusion genes arranged as closely spaced direct repeats in barley.

Ubiquitin (Ubi) genes encode two types of fusion proteins: polyUbi with a varying number of direct repeats of Ubi, and Ubi-tail fusions with long or short basic C-terminal extensions. A barley (Hordeum vulgare) genomic clone has been isolated with two very similar, intronless genes encoding monoUbi-long-tail fusion peptides. The genes are arranged as direct repeats separated by 3 kb of DNA and account for two of the probable three long-tail genes in the haploid barley genome. Both genes are active and give rise to messengers about 800 nt long. The sequence of the encoded Ubi moieties is identical to the sequence of Ubi repeats of polyUbi precursors from barley and other plants. The basic tails of the peptides are 79 aa long and 71-72% homologous to corresponding sequences from yeast and man. Recently, it was found that the long and short tails are ribosomal proteins in yeast [Finley et al., Nature 338 (1989) 394-401] and the evolutionary conservation of the structure of the Ubi-tail fusion genes suggests that they serve the same function in plants. The similarity between yeast and barley Ubi-long-tail fusion genes may extend to the regulatory regions, since upstream activating sites characteristic of ribosomal protein-encoding genes in yeast (UASrpg) were found in the barley genes.     

One-kilobase direct repeats of plasmid pSa

One-kilobase, direct repeats were found on either side of the chloramphenicol resistance gene of plasmid pSa. The right repeat corresponded to the region coding for sulfanilamide resistance. The repeats were not identical as judged by distances between restriction enzyme sites, hybridization, and by the ability to confer resistance to sulfanilamide.     

Multiple deletions of mtDNA remove the light strand origin of replication

Idiopathic inflammatory myopathies are progressive, debilitating muscle diseases. The pathogenesis of these disorders is multifactorial and appears to include mutations of the mitochondrial genome, which are usually indicated by morphological changes of mitochondria. The vast majority of all mitochondrial DNA deletions found are located between the origins of replication in the "major region" between nt5760-nt190. Using long distance PCR and sequencing techniques, we detected deletions which were unusually large (ca. 10500-12800 bp) and show uncommon 5'-breakpoints between nt800 and nt3326. Unlike most other deletions, their breakpoints are far upstream of the "major region." The atypical location of these deletions suggests a different pathomechanism. The impact of the mitochondrial DNA deletions in the pathogenetic cascade remains uncertain.     

Conformational mutation in human mtDNA detected by direct sequencing of enzymatically amplified DNA.

Restriction enzyme analysis of 241 human mtDNAs revealed polymorphism in the electrophoretic mobility of a fragment corresponding to part of the ND4 gene. Enzymatic amplification and direct sequencing of this fragment demonstrates that a single T--C transition correlates with the faster mobility exhibited by the fragment in seven mtDNAs from Papua New Guinea. The enhanced mobility caused by this transition could result from disrupting an AT-rich region near the middle of the fragment that might make it curve. An analogous mutation at an adjacent position in a European mtDNA causes a similar but more pronounced alteration in mobility. The seven New Guineans with this substitution are all from the Eastern Highlands Province and constitute one clade in a genealogical tree based upon restriction analysis. The additional information provided by sequencing allows refinement of the genealogical tree but does not require modification of higher order branching structure.     

Analysis of mtDNA variation in African populations reveals the most ancient of all human continent-specific haplogroups.

mtDNA sequence variation was examined in 140 Africans, including Pygmies from Zaire and Central African Republic (C.A.R.) and Mandenkalu, Wolof, and Pular from Senegal. More than 76% of the African mtDNAs (100% of the Pygmies and 67.3% of the Senegalese) formed one major mtDNA cluster (haplogroup L) defined by an African-specific HpaI site gain at nucleotide pair (np) 3592. Additional mutations subdivided haplogroup L into two subhaplogroups, each encompassing both Pygmy and Senegalese mtDNAs. A novel 12-bp homoplasmic insertion in the intergenic region between tRNA(Tyr) and cytochrome oxidase I (COI) genes was also observed in 17.6% of the Pygmies from C.A.R. This insertion is one of the largest observed in human mtDNAs. Another 25% of the Pygmy mtDNAs harbored a 9-bp deletion between the cytochrome oxidase II (COII) and tRNA(Lys) genes, a length polymorphism previously reported in non-African populations. In addition to haplogroup L, other haplogroups were observed in the Senegalese. These haplogroups were more similar to those observed in Europeans and Asians than to haplogroup L mtDNAs, suggesting that the African mtDNAs without the HpaI np 3592 site could be the ancestral types from which European and Asian mtDNAs were derived. Comparison of the intrapopulation sequence divergence in African and non-African populations confirms that African populations exhibit the largest extent of mtDNA variation, a result that further supports the hypothesis that Africans represent the most ancient human group and that all modern humans have a common and recent African origin. The age of the total African variation was estimated to be 101,000-133,000 years before present (YBP), while the age of haplogroup L was estimated at 98,000-130,000 YBP. These values substantially exceed the ages of all Asian- and European-specific mtDNA haplogroups.     

Two LINE 1 repeats in rat

One LINE 1 repeat has been located 661 bp downstream from the last albumin exon and another approx. 10 kbp downstream from the last alpha-fetoprotein exon in the rat genomic DNA. The LINE 1 repeat following the albumin gene is truncated at its 5' end and is 1204 nucleotides long. The 5' end of the longer repeat downstream from the alpha-fetoprotein gene has not been determined. The two repeats have 95% homology with each other, with the exception of a short diverse 3' end sequence just preceding the putative polyadenylation signal.     

5-Variable genome sequence of the LIVP vaccinia virus. A possible role for short direct repeats in the formation of deletions]

HindIII-O/N DNA fragments of vaccinia virus (VV) of the LIVP strain were mapped using thirteen restriction endonucleases. Nucleotide sequences of the HindIII-O fragment (1530 bp) as well as of a site of the HindIII-N genome fragment 353 bp in size were determined. Comparison of restriction maps and nucleotide sequences of VV strains (WR and LIVP) demonstrated that DNA of VV LIVP contained % deletions and 2 insertions. "Reliable" short direct repeats were localized and their possible role in formation of DNA deletions was shown. It was suggested that VV endonuclease and DNA-ligase participate in replication and repair processes. Mechanism of formation of variable sequences of viral genomes is discussed.     

Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin

Mitochondrial DNA (mtDNA) mutations and impaired respiratory function have been demonstrated in various tissues of aged individuals. We hypothesized that age-dependent increase of ROS and free radicals production in mitochondria is associated with the accumulation of large-scale mtDNA deletions. In this study, we first confirmed that the proportion of mtDNA with the 4977 bp deletion in human skin tissues increases with age. We then investigated the 8-hydroxy-2'-deoxyguanosine (8-OH-dG) content in skin tissues and lipid peroxides content of the skin fibroblasts from subjects of different ages. The results showed an age-dependent increase of 8-OH-dG level in the total DNA of skin tissues of the subjects above the age of 60 years. The specific content of malondialdehyde, an end product of lipid peroxidation, was also found to increase with age. On the other hand, we examined the enzyme activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPx) in the skin fibroblasts. The activities of Cu,Zn-SOD, catalase and glutathione peroxidase were found to decrease with age. However, the activity of Mn-SOD was increased with age before 60 years but was decreased thereafter. Moreover, the activity ratios of Mn-SOD/catalase and Mn-SOD/GPx exhibited the same pattern of change with age. This indicates that free radical scavenging enzymes can effectively dispose of ROS and free radicals before 60 years of age. However, elevated oxidative stress caused by an imbalance between the production and removal of ROS and free radicals occurred in skin fibroblasts after 60 years of age. Taken together, we suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process.     

mtDNA tandem repeats in domestic dogs and wolves: mutation mechanism studied by analysis of the sequence of imperfect repeats

The mitochondrial (mt) DNA control region (CR) of dogs and wolves contains an array of imperfect 10 bp tandem repeats. This region was studied for 14 domestic dogs representing the four major phylogenetic groups of nonrepetitive CR and for 5 wolves. Three repeat types were found among these individuals, distributed so that different sequences of the repeat types were formed in different molecules. This enabled a detailed study of the arrays and of the mutation events that they undergo. Extensive heteroplasmy was observed in all individuals; 85 different array types were found in one individual, and the total number of types was estimated at 384. Among unrelated individuals, no identical molecules were found, indicating a high rate of evolution of the region. By performing a pedigree analysis, array types which had been inherited from mother to offspring and array types which were the result of somatic mutations, respectively, could be identified, showing that about 20% of the molecules within an individual had somatic mutations. By direct pairwise comparison of the mutated and the original array types, the physiognomy of the inserted or deleted elements (indels) and the approximate positions of the mutations could be determined. All mutations could be explained by replication slippage or point mutations. The majority of the indels were 1-5 repeats long, but deletions of up to 17 repeats were found. Mutations were found in all parts of the arrays, but at a higher frequency in the 5' end. Furthermore, the inherited array types within the mother-offspring pair were aligned and compared so that germ line mutations could be studied. The pattern of the germ line mutations was approximately the same as that of the somatic mutations.     

Instability of large direct repeats in retrovirus vectors.

Retrovirus vectors were constructed with large (0.85- to 1.3-kilobase-pair) direct repeats in their genomes. Deletions involving the direct repeats occurred at a high frequency. Deletions occurred both when the direct repeats were in tandem and when they were separated by additional sequences. These deletions occurred during virus replication.