Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans

To safeguard genetic integrity, cells have evolved an accurate but not failsafe mechanism of DNA replication. Not all DNA sequences tolerate DNA replication equally well [1]. Also, genomic regions that impose structural barriers to the DNA replication fork are a potential source of genetic instability [2, 3]. Here, we demonstrate that G4 DNA-a sequence motif that folds into quadruplex structures in vitro [4, 5]-is highly mutagenic in vivo and is removed from genomes that lack dog-1, the C. elegans ortholog of mammalian FANCJ [6, 7], which is mutated in Fanconi anemia patients [8-11]. We show that sequences that match the G4 DNA signature G3-5N1-3G3-5N1-3G3-5N1-3G3-5 are deleted in germ and somatic tissues of dog-1 animals. Unbiased aCGH analyses of dog-1 genomes that were allowed to accumulate mutations in >100 replication cycles indicate that deletions are found exclusively at G4 DNA; deletion frequencies can reach 4% per site per animal generation. We found that deletion sizes fall short of Okazaki fragment lengths [12], and no significant microhomology was observed at deletion junctions. The existence of 376,000 potentially mutagenic G4 DNA sites in the human genome could have major implications for the etiology of hereditary FancJ and nonhereditary cancers.     

LINE drive. retrotransposition and genome instability

The LINE-1 (L1) retrotransposon, the most important human mobile element, shapes the genome in many ways. Now two groups provide evidence that L1 retrotransposition is associated with large genomic deletions and inversions in transformed cells. If these events occur at a similar frequency in vivo, they have had a substantial effect on human genome evolution.     

Combing the genome for genomic instability

Genomic instability is one of the major features of cancer cells. The clinical phenotypes associated with several human diseases have been linked to recurrent DNA rearrangements and dysfunction of DNA replication processes that involve unstable genomic regions. Analysis of these rearrangements, which are frequently submicroscopic and can lead to loss or gain of dosage-sensitive genes or gene disruption, requires the development of sensitive, high-resolution techniques. This will lead to a better understanding of the mechanisms underlying genome instability and a greater awareness of the role of chromosomal rearrangements in disease. A new technology that involves molecular combing, a method that permits straightening and aligning molecules of genomic DNA, should make possible a detailed analysis of genomic events at the level of single DNA molecules. Such a single molecule approach could help to elucidate important properties that are masked in bulk studies.     

Mitochondrial genome instability in human cancers

Malfunction of mismatch repair (MMR) genes produces nuclear genome instability (NGI) and plays an important role in the origin of some hereditary and sporadic human cancers. The appearance of non-inherited microsatellite alleles in tumor cells (microsatellite instability, MSI) is one of the expressions of NGI. We present here data showing mitochondrial genome instability (mtGI) in most of the human cancers analyzed so far. The mtDNA markers used were point mutations, length-tract instability of mono- or dinucleotide repeats, mono- or dinucleotide insertions or deletions, and long deletions. Comparison of normal and tumoral tissues from the same individual reveals that mt-mutations may show as homoplasmic (all tumor cells have the same variant haplotype) or as heteroplasmic (tumor cells are a mosaic of inherited and acquired variant haplotypes). Breast, colorectal, gastric and kidney cancers exhibit mtGI with a pattern of mt-mutations specific for each tumor. No correlation between NGI and mtGI was found in breast, colorectal or kidney cancers, while a positive correlation was found in gastric cancer. Conversely, germ cell testicular cancers lack mtGI. Damage by reactive oxygen species (ROS), slipped-strand mispairing (SSM) and deficient repair are the causes explaining the appearance of mtGI. The replication and repair of mtDNA are controlled by nuclear genes. So far, there is no clear evidence linking MMR gene malfunction with mtGI. Polymerase gamma (POLgamma) carries out the mtDNA synthesis. Since this process is error-prone due to a deficiency in the proofreading activity of POLgamma, this enzyme has been assumed to be involved in the origin of mt-mutations. Somatic cells have hundreds to thousands of mtDNA molecules with a very high rate of spontaneous mutations. Accordingly, most somatic cells probably have a low frequency of randomly mutated mtDNA molecules. Most cancers are of monoclonal origin. Hence, to explain the appearance of mtGI in tumors we have to explain why a given variant mt-haplotype expands and replaces part of (heteroplasmy) or all (homoplasmy) wild mt-haplotypes in cancer cells. Selective and/or replicative advantage of some mutations combined with a severe bottleneck during the mitochondrial segregation accompanying mitosis are the mechanisms probably involved in the origin of mtGI.     

Telomere dysfunction in genome instability syndromes

Telomeres are nucleoprotein complexes located at the end of eukaryotic chromosomes. They have essential roles in preventing terminal fusions, protecting chromosome ends from degradation, and in chromosome positioning in the nucleus. These terminal structures consist of a tandemly repeated DNA sequence (TTAGGG in vertebrates) that varies in length from 5 to 15 kb in humans. Several proteins are attached to this telomeric DNA, some of which are also involved in different DNA damage response pathways, including Ku80, Mre11, NBS and BLM, among others. Mutations in the genes encoding these proteins cause a number of rare genetic syndromes characterized by chromosome and/or genetic instability and cancer predisposition. Deletions or mutations in any of these genes may also cause a telomere defect resulting in accelerated telomere shortening, lack of end-capping function, and/or end-to-end chromosome fusions. This telomere phenotype is also known to promote chromosomal instability and carcinogenesis. Therefore, it is essential to understand the interplay between telomere biology and genome stability. This review is focused in the dual role of chromosome fragility proteins in telomere maintenance.     

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.     

Stress-induced modulators of repeat instability and genome evolution

Evolution hinges on the ability of organisms to adapt to their environment. A key regulator of adaptability is mutation rate, which must be balanced to maintain genome fidelity while permitting sufficient plasticity to cope with environmental changes. Multiple mechanisms govern an organism's mutation rate. Constitutive mechanisms include mutator alleles that drive global, permanent increases in mutation rates, but these changes are confined to the subpopulation that carries the mutator allele. Other mechanisms focus mutagenesis in time and space to improve the chances that adaptive mutations can spread through the population. For example, environmental stress can induce mechanisms that transiently relax the fidelity of DNA repair to bring about a temporary increase in mutation rates during times when an organism experiences a reduced fitness for its surroundings, as has been demonstrated for double-strand break repair in Escherichia coli. Still, other mechanisms control the spatial distribution of mutations by directing changes to especially mutable sequences in the genome. In eukaryotic cells, for example, the stress-sensitive chaperone Hsp90 can regulate the length of trinucleotide repeats to fine-tune gene function and can regulate the mobility of transposable elements to enable larger functional changes. Here, we review the regulation of mutation rate, with special emphasis on the roles of tandem repeats and environmental stress in genome evolution.     

DNA replication stress, genome instability and aging

Genome instability is a fundamentally important component of aging in all eukaryotes. How age-related genome instability occurs remains unclear. The free radical theory of aging posits oxidative damage to DNA and other cellular constituents as a primary determinant of aging. More recent versions of this theory predict that mitochondria are a major source of reactive oxygen species (ROS) that cause oxidative damage. Although substantial support for the free radical theory exists, the results of some tests of this theory have been contradictory or inconclusive. Enhanced growth signaling also has been implicated in aging. Many efforts to understand the effects of growth signaling on aging have focused on inhibition of oxidative stress responses that impact oxidative damage. However, recent experiments in the model organism Saccharomyces cerevisiae (budding yeast) and in higher eukaryotes suggest that growth signaling also impacts aging and/or age-related diseases—including cancer and neurodegeneration—by inducing DNA replication stress, which causes DNA damage. Replication stress, which has not been broadly considered as a factor in aging, may be enhanced by ROS that signal growth. In this article, we review evidence that points to DNA replication stress and replication stress-induced genome instability as important factors in aging.     

Mechanisms of genome instability in Hutchinson-Gilford progeria

Background

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder. It arises from a single point mutation in the LMNA gene. This mutation stimulates an aberrant splicing event and produces progerin, an isoform of the lamin A protein. Accumulation of progerin disrupts numerous physiological pathways and induces defects in nuclear architecture, gene expression, histone modification, cell cycle regulation, mitochondrial functionality, genome integrity and much more.

Objective

Among these phenotypes, genomic instability is tightly associated with physiological aging and considered a main contributor to the premature aging phenotypes. However, our understanding of the underlying molecular mechanisms of progerin-caused genome instability is far from clear.

Results and Conclusion

In this review, we summarize some of the recent findings and discuss potential mechanisms through which, progerin affects DNA damage repair and leads to genome integrity.

     

From polyploidy to aneuploidy, genome instability and cancer

Polyploidy is a frequent phenomenon in the eukaryotic world, but the biological properties of polyploid cells are not well understood. During evolution, polyploidy is thought to be an important mechanism that contributes to speciation. Polyploid, usually non-dividing, cells are formed during development in otherwise diploid organisms. A growing amount of evidence indicates that polyploid cells also arise during a variety of pathological conditions. Genetic instability in these cells might provide a route to aneuploidy and thereby contribute to the development of cancer.     

DNA damage tolerance,mismatch repair and genome instability

DNA mismatch repair is an important pathway of mutation avoidance. It also contributes to the cytotoxic effects of some kinds of DNA damage, and cells defective in mismatch repair are resistant, or tolerant, to the presence of some normally cytotoxic base analogues in their DNA. The absence of a particular mismatch binding function from some mammalian cells confers resistance to the base analogues O⁶-methylguanine and 6-thioguanine in DNA. Cells also acquire a spontaneous mutator phenotype as a consequence of this defect. Impaired mismatch binding can cause an instability in DNA microsatellite regions that comprise repeated dinucleotides. Microsatellite DNA instability is common in familial and sporadic colon carcinomas as well as in a number of other tumours. Several independent lines of investigation have identified defects in mismatch repair proteins that are causally related to these cancers.     

Aberrant genome size and instability of Phytophthora ramorum oospore progenies

The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogen's ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements.     

Links between replication, recombination and genome instability in eukaryotes

Double-strand breaks in DNA can be repaired by homologous recombination including break-induced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.