Analysis of phylogenetically reconstructed mutational spectra in human mitochondrial DNA control region

Analysis of mutations in mitochondrial DNA is an important issue in population and evolutionary genetics. To study spontaneous base substitutions in human mitochondrial DNA we reconstructed the mutational spectra of the hypervariable segments I and II (HVS I and II) using published data on polymorphisms from various human populations. An excess of pyrimidine transitions was found both in HVS I and II regions. By means of classification analysis numerous mutational hotspots were revealed in these spectra. Context analysis of hotspots revealed a complex influence of neighboring bases on mutagenesis in the HVS I region. Further statistical analysis suggested that a transient misalignment dislocation mutagenesis operating in monotonous runs of nucleotides play an important role for generating base substitutions in mitochondrial DNA and define context properties of mtDNA. Our results suggest that dislocation mutagenesis in HVS I and II is a fingerprint of errors produced by DNA polymerase gamma in the course of human mitochondrial DNA replication     

A preliminary analysis of the DNA and diet of the extinct Beothuk: a systematic approach to ancient human DNA

We have used a systematic protocol for extracting, quantitating, sexing and validating ancient human mitochondrial and nuclear DNA of one male and one female Beothuk, a Native American population from Newfoundland, which became extinct approximately 180 years ago. They carried mtDNA haplotypes, which fall within haplogroups X and C, consistent with Northeastern Native populations today. In addition we have sexed the male using a novel-sexing assay and confirmed the authenticity of his Y chromosome with the presence of the Native American specific Y-QM3 single nucleotide polymorphism (SNP). This is the first ancient nuclear SNP typed from a Native population in the Americas. In addition, using the same teeth we conducted a stable isotopes analysis of collagen and dentine to show that both individuals relied on marine sources (fresh and salt water fish, seals) with no hierarchy seen between them, and that their water sources were pooled or stored water. Both mtDNA sequence data and Y SNP data hint at possible gene flow or a common ancestral population for both the Beothuk and the current day Mikmaq, but more importantly the data do not lend credence to the proposed idea that the Beothuk (specifically, Nonosabasut) were of admixed (European-Native American) descent. We also analyzed patterns of DNA damage in the clones of authentic mtDNA sequences; there is no tendency for DNA damage to occur preferentially at previously defined mutational hotspots, suggesting that such mutational hotspots are not hypervariable because they are more prone to damage.     

Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10⁻⁸ per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.     

Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.     

How can we distinguish between mutational "hot spots" and "old sites" in human mtDNA samples?

New research into variation in mutation rates across nucleotide positions in human mitochondrial DNA (mtDNA) calls into question population genetics models that assume a constant mutation rate for all sites in a sequence, particularly for hypervariable control region segments I and II. Related to this research is discovering the extent to which highly polymorphic sites are really mutational "hot spots" rather than "old" sites rooted early in the phylogenetic tree. This issue is addressed through the analysis of linkage disequilibrium patterns in the mtDNAs of 10 human populations. Hot spots can be expected to show little or no disequilibrium since they can be interpreted as having randomly expressed patterns. In fact, the results suggest that many highly polymorphic sites are not old sites, but instead are hot spots. Suspected hot spots are listed and compared with hypervariable sites given by Wakeley (1993) and Hasegawa et al. (1993).     

Variation of Human Mitochondrial DNA: Distribution of Hot Spots in Hypervariable Segment I of the Major Noncoding Region

Mitochondrial DNA (mtDNA) samples belonging to fifteen phylogenetically related mtDNA types specific to the populations of Europe (H, V, J, T, U, K, I, W, and X) and Northern Asia (A, C, D, G, Y, and Z) were typed for sequence variation in hypervariable segment I (HVSI). The approach used allowed to distinguish several hypervariable sites at nucleotide positions 16093, 16129, 16189, 16311, and 16362. Identical mutations at these sites were found in 10–11 out of 15 mtDNA groups examined. Positions 16126, 16172, 16192, 16256, 16261, 16291, 16293, and 16298 appeared to be less variable, since parallel mutations at these sites were found in 6–8 European and Asian mtDNA groups. The examples of the effects of mutations in hypervariable positions at the major noncoding mtDNA region on the frequency of reverse mutations in other mtDNA regions are presented. It was shown that such effects of nucleotide context on the mutation rate could be observed in phylogenetic mtDNA networks such as cyclic structures like rhombs and cubes. Analogous structures in the networks could be seen also in the case of the appearance of recombinant mtDNA types resulted from homologous recombination between mtDNA molecules in heteroplasmic mixture. The problem of the effect of polynucleotide context on the intensity of mtDNA mutagenesis is discussed. Recombination processes along with site-directed mutagenesis caused by action of genetic factors (of nuclear genome) and/or of the environment are considered as possible mechanisms of mitochondrial genome evolution.     

Variation of human mitochondrial DNA: distribution of hot spots in hypervariable segment I of the major noncoding region

Mitochondrial DNA (mtDNA) samples belonging to fifteen phylogenetically related mtDNA types specific to the populations of Europe (H, V, J, T, U, K, I, W, and X) and Northern Asia (A, C, D, G, Y, and Z) were typed for sequence variation in hypervariable segment I (HVSI). The approach used allowed to distinguish several hypervariable sites at nucleotide positions 16093, 16129, 16189, 16311, and 16362. Identical mutations at these sites were found in 10-11 out of 15 mtDNA groups examined. Positions 16126, 16172, 16192, 16256, 16261, 16291, 16293, and 16298 appeared to be less variable, since parallel mutations at these sites were found in 6-8 European and Asian mtDNA groups. The examples of the effects of mutations in hypervariable positions at the major noncoding mtDNA region on the frequency of reverse mutations in other mtDNA regions are presented. It was shown that such effects of nucleotide context on the mutation rate could be observed in phylogenetic mtDNA networks such as cyclic structures like rhombs and cubes. Analogous structures in the networks could be seen also in the case of the appearance of recombinant mtDNA types resulted from homologous recombination between mtDNA molecules in heteroplasmic mixture. The problem of the effect of polynucleotide context on the intensity of mtDNA mutagenesis is discussed. Recombination processes along with site-directed mutagenesis caused by action of genetic factors (of nuclear genome) and/or of the environment are considered as possible mechanisms of mitochondrial genome evolution.     

Hypervariable noncoding sequences in Saccharomyces cerevisiae

Compared to protein-coding sequences, the evolution of noncoding sequences and the selective constraints placed on these sequences is not well characterized. To compare the evolution of coding and noncoding sequences, we have conducted a survey for DNA polymorphism at five randomly chosen loci among a diverse collection of 81 strains of Saccharomyces cerevisiae. Average rates of both polymorphism and divergence are 40% lower at noncoding sites and 90% lower at nonsynonymous sites in comparison to synonymous sites. Although noncoding and coding sequences show substantial variability in ratios of polymorphism to divergence, two of the loci, MLS1 and PDR10, show a higher rate of polymorphism at noncoding compared to synonymous sites. The high rate of polymorphism is not accompanied by a high rate of divergence and is limited to a few small regions. These hypervariable regions include sites with three segregating bases at a single site and adjacent polymorphic sites. We show that this clustering of polymorphic sites is significantly greater than one would expect on the basis of the spacing between polymorphic fourfold degenerate sites. Although hypervariable noncoding sequences could result from selection on regulatory mutations, they could also result from transient mutational hotspots.     

Median networks: speedy construction and greedy reduction, one simulation, and two case studies from human mtDNA

Molecular data sets characterized by few phylogenetically informative characters with a broad spectrum of mutation rates, such as intraspecific control-region sequence variation of human mitochondrial DNA (mtDNA), can be usefully visualized in the form of median networks. Here we provide a step-by-step guide to the construction of such networks by hand. We improve upon a previously implemented algorithm by outlining an efficient parametrized strategy amenable to large data sets, greedy reduction, which makes it possible to reconstruct some of the confounding recurrent mutations. This entails some postprocessing as well, which assists in capturing more parsimonious solutions. To simplify the creation of the resulting network by hand, we describe a speedy approach to network construction, based on a careful planning of the processing order. A coalescent simulation tailored to human mtDNA variation in Eurasia testifies to the usefulness of reduced median networks, while highlighting notorious problems faced by all phylogenetic methods in this context. Finally, we discuss two case studies involving the comparison of characters in the two hypervariable segments of the human mtDNA control region in the light of the worldwide control-region sequence database, as well as additional restriction fragment length polymorphism information. We conclude that only a minority of the mutations that hit the second segment occur at sites that would have a mutation rate comparable to those at most sites in the first segment. Discarding the known "noisy" sites of the second segment enhances the analysis.     

Biophysical simulations and structure-based modeling of residue interaction networks in the tumor suppressor proteins reveal functional role of cancer mutation hotspots in molecular communication

In the current study, we have combined molecular simulations and energetic analysis with dynamics-based network modeling and perturbation response scanning to determine molecular signatures of mutational hotspot residues in the p53, PTEN, and SMAD4 tumor suppressor proteins. By examining structure, energetics and dynamics of these proteins, we have shown that inactivating mutations preferentially target a group of structurally stable residues that play a fundamental role in global propagation of dynamic fluctuations and mediating allosteric interaction networks. Through integration of long-range perturbation dynamics and network-based approaches, we have quantified allosteric potential of residues in the studied proteins. The results have revealed that mutational hotspot sites often correspond to high centrality mediating centers of the residue interaction networks that are responsible for coordination of global dynamic changes and allosteric signaling. Our findings have also suggested that structurally stable mutational hotpots can act as major effectors of allosteric interactions and mutations in these positions are typically associated with severe phenotype. Modeling of shortest inter-residue pathways has shown that mutational hotspot sites can also serve as key mediating bridges of allosteric communication in the p53 and PTEN protein structures. Multiple regression models have indicated that functional significance of mutational hotspots can be strongly associated with the network signatures serving as robust predictors of critical regulatory positions responsible for loss-of-function phenotype. The results of this computational investigation are compared with the experimental studies and reveal molecular signatures of mutational hotspots, providing a plausible rationale for explaining and localizing disease-causing mutations in tumor suppressor genes.     

Detecting Heteroplasmy from High-Throughput Sequencing of Complete Human Mitochondrial DNA Genomes

Heteroplasmy, the existence of multiple mtDNA types within an individual, has been previously detected by using mostly indirect methods and focusing largely on just the hypervariable segments of the control region. Next-generation sequencing technologies should enable studies of heteroplasmy across the entire mtDNA genome at much higher resolution, because many independent reads are generated for each position. However, the higher error rate associated with these technologies must be taken into consideration to avoid false detection of heteroplasmy. We used simulations and phiX174 sequence data to design criteria for accurate detection of heteroplasmy with the Illumina Genome Analyzer platform, and we used artificial mixtures and replicate data to test and refine the criteria. We then applied these criteria to mtDNA sequence reads for 131 individuals from five Eurasian populations that had been generated via a parallel tagged approach. We identified 37 heteroplasmies at 10% frequency or higher at 34 sites in 32 individuals. The mutational spectrum does not differ between heteroplasmic mutations and polymorphisms in the same individuals, but the relative mutation rate at heteroplasmic mutations is significantly higher than that estimated for all mutable sites in the human mtDNA genome. Moreover, there is also a significant excess of nonsynonymous mutations observed among heteroplasmies, compared to polymorphism data from the same individuals. Both mutation-drift and negative selection influence the fate of heteroplasmies to determine the polymorphism spectrum in humans. With appropriate criteria for avoiding false positives due to sequencing errors, next-generation technologies can provide novel insights into genome-wide aspects of mtDNA heteroplasmy.     

Mutagenic specificity of ultraviolet light

Genetic and sequencing studies of ultraviolet light (u.v.)-induced mutations in the lacI gene of Escherichia coli show the following: u.v. stimulates many types of mutations. In lacI, base substitutions account for 60 to 65% of the observed mutations, small frameshifts 30 to 35%, and deletions of more than several base-pairs approximately 5%. A comparison of the mutational spectrum of u.v.-induced mutations with those of other SOS-dependent mutagens and with the mutations produced by inducing the SOS system in the absence of mutagenic treatment indicates that most u.v.-induced base substitutions are "targeted", resulting from premutational lesions across from the site of the mutations. Among base substitutions, both transitions and transversions occur, although the most favored mutational sites involve G X C----A X T transitions. G X C----A X T transitions are induced preferentially at sites of adjacent pyrimidines. In one case the conversion of a site from -A-C-A- to -T-C-A- results in a 15-fold increase in u.v.-induced C----T transitions. Frameshifts at certain sites are well-induced by u.v., and the largest hotspot in the I gene involves the loss of an (sequence in text) base pair from a (sequence in text) sequence. Of 25 frameshifts detected by DNA sequencing, 23 mutations at seven different sites result from the elimination of a single base-pair, and two mutations result from the elimination of two base-pairs. No additions were detected. The use of a lacI-Z fusion system, which allows direct selection of frameshifts of either sign, reveals that throughout the entire gene frameshifts that eliminate a single base-pair (-1) predominate by a factor of 20 or more over frameshifts that add a single base-pair (+1). In one case a two-base-pair elimination occurs frequently, resulting in the loss of a -C-T- sequence (on one strand), or a -T-C- sequence, from a -C-T-C-T-C-T-C- sequence. For both frameshifts and base substitutions, some aspect of the larger surrounding sequence beyond the nearest neighbors can influence mutation rates by as much as 50-fold, thus determining which sites are seen as hotspots. The bearing of these and other data on the detailed mechanism of mutagenesis is considered in the Discussion.     

Low mutational burden of individual acquired mitochondrial DNA mutations in brain

Neurons may be particularly susceptible to oxidative damage, which has been proposed to induce somatic mutations, particularly in mitochondrial DNA (mtDNA). Therefore, acquired mtDNA mutations might preferentially accumulate in the brain and could play a role in aging and neurodegenerative disorders. Recently, a somatic T to G mtDNA mutation at noncoding nucleotide position 414 was reported in fibroblasts specifically from elderly subjects, with mutational burdens of up to 50%. We screened for this mutation in brain-derived mtDNA from 8 Alzheimer's disease patients, 27 Parkinson's disease patients, 4 multiple system atrophy patients, and 44 controls using up to three RFLP analyses. A total of 73 of these subjects were over the age of 65. The 414 mutation was absent in all cases. Next, individual mtDNA fragments from 6 elderly subjects were cloned, and a total of 70 clones were sequenced. The 414 mutation was absent in all clones, though occasional sequence variations were identified at other sites in single clones. The 414 mutation also was absent in blood (n = 6) and fibroblasts (n = 11) from elderly subjects. Our data suggest that it is rare for any one particular acquired mtDNA mutation to reach levels in the brain that are functionally significant. This does not exclude the possibility that the cumulative burden of multiple, individually rare, acquired mutations impairs mitochondrial function.     

Phylogenetic and familial estimates of mitochondrial substitution rates: study of control region mutations in deep-rooting pedigrees

We studied mutations in the mtDNA control region (CR) using deep-rooting French-Canadian pedigrees. In 508 maternal transmissions, we observed four substitutions (0.0079 per generation per 673 bp, 95% CI 0.0023-0.186). Combined with other familial studies, our results add up to 18 substitutions in 1,729 transmissions (0.0104), confirming earlier findings of much greater mutation rates in families than those based on phylogenetic comparisons. Only 12 of these mutations occurred at independent sites, whereas three positions mutated twice each, suggesting that pedigree studies preferentially reveal a fraction of highly mutable sites. Fitting the data through use of a nonuniform rate model predicts the presence of 40 (95% CI 27-54) such fast sites in the whole CR, characterized by the mutation rate of 274 per site per million generations (95% CI 138-410). The corresponding values for hypervariable regions I (HVI; 1,729 transmissions) and II (HVII; 1,956 transmissions), are 19 and 22 fast sites, with rates of 224 and 274, respectively. Because of the high probability of recurrent mutations, such sites are expected to be of no or little informativity for the evaluation of mutational distances at the phylogenetic time scale. The analysis of substitution density in the alignment of 973 HVI and 650 HVII unrelated European sequences reveals that the bulk of the sites mutate at relatively moderate and slow rates. Assuming a star-like phylogeny and an average time depth of 250 generations, we estimate the rates for HVI and HVII at 23 and 24 for the moderate sites and 1.3 and 1.0 for the slow sites. The fast, moderate, and slow sites, at the ratio of 1:2:13, respectively, describe the mutation-rate heterogeneity in the CR. Our results reconcile the controversial rate estimates in the phylogenetic and familial studies; the fast sites prevail in the latter, whereas the slow and moderate sites dominate the phylogenetic-rate estimations.     

High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

Background

Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL.

Methodology/Principal Findings

The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis.

Conclusions/Significance

Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis.     

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational ‘hot-spots’ or ‘cold-spots’. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.     

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

Involvement of 5-methylcytosine in sunlight-induced mutagenesis.

In human skin cancers, more than 30 % of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. In Xorder to define the contribution of 5-methylcytosine to sunlight-induced mutations, we have used mouse fibroblasts containing the CpG-methylated lacI transgene as a mutational target. We sequenced 182 UVC (254 nm UV)-induced mutations and 170 mutations induced by a solar UV simulator, along with 75 mutations in untreated cells. Only a few of the mutations in untreated cells were transitions at dipyrimidines, but more than 95% of the UVC and solar irradiation-induced mutations were targeted to dipyrimidine sites, the majority being transitions. After UVC irradiation, 6% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only 2.2% of all mutations were transitions within this sequence context. However, 24% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Two sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form the highest levels of cyclobutane pyrimidine dimers after irradiation with sunlight but not with UVC. The data indicate that dipyrimidines that contain 5-methylcytosine are preferential targets for sunlight-induced mutagenesis in cultured mammalian cells, thus explaining the large proportion of p53 mutations at such sites in skin tumors in vivo.     

Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees

The mitochondrial DNA (mtDNA) of 87 index cases with Leber hereditary optic neuropathy (LHON) sequentially diagnosed in Italy, including an extremely large Brazilian family of Italian maternal ancestry, was evaluated in detail. Only seven pairs and three triplets of identical haplotypes were observed, attesting that the large majority of the LHON mutations were due to independent mutational events. Assignment of the mutational events into haplogroups confirmed that J1 and J2 play a role in LHON expression but narrowed the association to the subclades J1c and J2b, thus suggesting that two specific combinations of amino acid changes in the cytochrome b are the cause of the mtDNA background effect and that this may occur at the level of the supercomplex formed by respiratory-chain complexes I and III. The families with identical haplotypes were genealogically reinvestigated, which led to the reconnection into extended pedigrees of three pairs of families, including the Brazilian family with its Italian counterpart. The sequencing of entire mtDNA samples from the reconnected families confirmed the genealogical reconstruction but showed that the Brazilian family was heteroplasmic at two control-region positions. The survey of the two sites in 12 of the Brazilian subjects revealed triplasmy in most cases, but there was no evidence of the tetraplasmy that would be expected in the case of mtDNA recombination.     

8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand.