Non-B DNA structure-induced genetic instability

Repetitive DNA sequences are abundant in eukaryotic genomes, and many of these sequences have the potential to adopt non-B DNA conformations. Genes harboring non-B DNA structure-forming sequences increase the risk of genetic instability and thus are associated with human diseases. In this review, we discuss putative mechanisms responsible for genetic instability events occurring at these non-B DNA structures, with a focus on hairpins, left-handed Z-DNA, and intramolecular triplexes or H-DNA. Slippage and misalignment are the most common events leading to DNA structure-induced mutagenesis. However, a number of other mechanisms of genetic instability have been proposed based on the finding that these structures not only induce expansions and deletions, but can also induce DNA strand breaks and rearrangements. The available data implicate a variety of proteins, such as mismatch repair proteins, nucleotide excision repair proteins, topoisomerases, and structure specific-nucleases in the processing of these mutagenic DNA structures. The potential mechanisms of genetic instability induced by these structures and their contribution to human diseases are discussed.     

Precision genetic engineering in large mammals

Precision genetic engineering based on stable chromosomal insertion of exogenous DNA in the genomes of large mammals is immensely important for the development of improved biomedical models, pharmaceutical research and an accelerated breeding progress. Precision genetic engineering requires (i) a known locus of genomic integration, (ii) a defined status of foreign DNA, (iii) that transgene expression is unaffected by neighbouring chromosomal sequences, (iv) endogenous genes are not mutated and (v) no unwanted DNA sequences are present. Recently, advanced molecular techniques exploiting exogenous enzymes have opened the possibilities for more sophisticated genetic engineering. Here, we critically review current developments of enzyme-catalysed approaches for targeted transgenesis in large mammals.     

哺乳动物中的DNA主动去甲基化

DNA甲基化是一种相对稳定且可遗传的表观遗传标记,在植物和动物细胞中均发现有DNA主动去甲基化现象,其机制在植物中已基本得到阐释,但在哺乳动物中尚未鉴定出一种有效的DNA去甲基化酶,并且DNA主动去甲基化途径也存在争议。文章综合分析了近期的文献资料,阐述了哺乳动物中发生DNA主动去甲基化的时空特异性,并从细胞和组织特异性角度介绍DNA主动去甲基化的可能通路和机制,即5-甲基胞嘧啶的氧化作用、5-甲基胞嘧啶脱氨基以及DNA修复等,旨在为破译表观遗传重编程过程提供理论依据。     

Genome-wide DNA demethylation in mammals

The cytidine deaminase AID and elongator-complex proteins contribute to the extensive removal of DNA methylation in mammalian primordial germ cells and in the paternal pronucleus of the zygote.     

Transposable elements controlling genetic instabilities in mammals

It is proposed that the instabilities in gene action of some alleles at certain loci in the mouse (e.g., a, c, H-2 Mi, p, pe, T, Va, W), which do not seem to conform to traditional hypotheses of gene action, are better interpretable in the light of modern studies of transposable DNA elements (insertion sequences and transposons of prokaryotic organisms; controlling elements of maize; transposable controlling elements of Drosophila). Some phenotypic evidence in the mouse in support of this hypothesis is presented for the a, Mi, p, and W loci, which affect pigmentation.     

Determination of mitochondrial genetic diversity in mammals

Mitochondrial DNA (mtDNA) is one of the most popular population genetic markers. Its relevance as an indicator of population size and history has recently been questioned by several large-scale studies in animals reporting evidence for recurrent adaptive evolution, at least in invertebrates. Here we focus on mammals, a more restricted taxonomic group for which the issue of mtDNA near neutrality is crucial. By analyzing the distribution of mtDNA diversity across species and relating it to allozyme diversity, life-history traits, and taxonomy, we show that (i) mtDNA in mammals does not reject the nearly neutral model; (ii) mtDNA diversity, however, is unrelated to any of the 14 life-history and ecological variables that we analyzed, including body mass, geographic range, and The World Conservation Union (IUCN) categorization; (iii) mtDNA diversity is highly variable between mammalian orders and families; (iv) this taxonomic effect is most likely explained by variations of mutation rate between lineages. These results are indicative of a strong stochasticity of effective population size in mammalian species. They suggest that, even in the absence of selection, mtDNA genetic diversity is essentially unpredictable, knowing species biology, and probably uncorrelated to species abundance.     

DNA structure-induced recruitment and activation of the Fanconi anemia pathway protein FANCD2

The Fanconi anemia (FA) pathway proteins are thought to be involved in the repair of irregular DNA structures including those encountered by the moving replication fork. However, the nature of the DNA structures that recruit and activate the FA proteins is not known. Because FA proteins function within an extended network of proteins, some of which are still unknown, we recently established cell-free assays in Xenopus laevis egg extracts to deconstruct the FA pathway in a fully replication-competent context. Here we show that the central FA pathway protein, xFANCD2, is monoubiquitinated (xFANCD2-L) rapidly in the presence of linear and branched double-stranded DNA (dsDNA) structures but not single-stranded or Y-shaped DNA. xFANCD2-L associates with dsDNA structures in an FA core complex-dependent manner but independently of xATRIP, the regulatory subunit of xATR. Formation of xFANCD2-L is also triggered in response to circular dsDNA, suggesting that dsDNA ends are not required to trigger monoubiquitination of FANCD2. The induction of xFANCD2-L in response to circular dsDNA is replication and checkpoint independent. Our results provide new evidence that the FA pathway discriminates among DNA structures and demonstrate that triggering the FA pathway can be uncoupled from DNA replication and ATRIP-dependent activation.     

Aging and genetic instability in yeast

There is a striking link between increasing age and the incidence of cancer in humans. One of the hallmarks of cancer, genomic instability, has been observed in all types of organisms. In the yeast Saccharomyces cerevisiae, it was recently discovered that during the replicative lifespan, aging cells switch to a state of high genomic instability that persists until they die. In considering these and other recent results, we suggest that accumulation of oxidatively damaged protein in aging cells results in the loss of function of gene products critical for maintaining genome integrity. Determining the identity of these proteins and how they become damaged represents a new challenge for understanding the relationship between age and genetic instability.     

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.     

DNA methylation and demethylation in mammals

Cell type-specific DNA methylation patterns are established during mammalian development and maintained in adult somatic cells. Understanding how these patterns of 5-methylcytosine are established and maintained requires the elucidation of mechanisms for both DNA methylation and demethylation. The enzymes involved in the de novo methylation of DNA and the maintenance of the resulting methylation patterns have been fairly well characterized. However, important remaining challenges are to understand how DNA methylation systems function in vivo and in the context of chromatin. In addition, the enzymes and mechanisms for demethylation remain to be elucidated. There is still no consensus as to how active enzymatic demethylation is achieved in mammalian cells, but recent studies implicate base excision repair for genome-wide DNA demethylation in germ cells and early embryos.     

Long DNA palindromes,cruciform structures,genetic instability and secondary structure repair

Long DNA palindromes pose a threat to genome stability. This instability is primarily mediated by slippage on the lagging strand of the replication fork between short directly repeated sequences close to the ends of the palindrome. The role of the palindrome is likely to be the juxtaposition of the directly repeated sequences by intrastrand base-pairing. This intra-strand base-pairing, if present on both strands, results in a cruciform structure. In bacteria, cruciform structures have proved difficult to detect in vivo, suggesting that if they form, they are either not replicated or are destroyed. SbcCD, a recently discovered exonuclease of Escherichia coli, is responsible for preventing the replication of long palindromes. These observations lead to the proposal that cells may have evolved a post-replicative mechanism for the elimination and/or repair of large DNA secondary structures.     

Adaptation to DNA damage and stimulation of genetic instability: the double-edged sword mammalian DNA polymerase kappa

A major tolerance mechanism that functions to replicate damaged genomic DNA across lesions that have escaped elimination by repair mechanism is translesion DNA synthesis (TLS). DNA polymerase kappa (Pol kappa), a specialised low-fidelity DNA polymerase which is able to perform DNA synthesis across several damaged bases, is one of the enzymes involved in the process. The mutagenic nature of Pol kappa implies that its expression must be tightly regulated to prevent the formation of excessive genetic disorders along undamaged parts of the genome. Indeed, Pol kappa overexpression, which is notably observed in lung cancer, results not only in increased spontaneous mutagenesis, but also in pleiotropic alterations such as DNA breaks, genetic exchanges and aneuploidy. This review will discuss both aspects of DNA polymerase kappa, which can be considered as a genomic supervisor participating in genome maintenance and when misregulated as a genetic instability enhancer as well.     

Oncovirus-induced permanent genetic instability in Drosophila melanogaster

Mutant alleles of a system of genetic instability induced by oncoviral DNAs were shown to demonstrate an unstable manifestation 500 generations after their emergence. A cytogenetic analysis of oncovirus-induced unstable lines has revealed numerous chromosome rearrangements. For the Lobe alleles of this system, a specific chromosome rearrangement, Df(2L) = 35C-36B, was found on the left arm of chromosome 2. We used recessive lethal mutations involving DNA rearrangements in a successful construction of cross systems for "explosive" instability.     

The effect of inhibitors of DNA repair on the genetic instability of Streptomyces cattleya

Various streptomycetes show well defined instabilities that do not appear to be attributable to plasmid loss. The unstable phenotype, in many cases, arises at frequencies too high to be explained by point mutations. The frequency of instability can be enhanced by UV irradiation. Two major repair systems have been found in Escherichia coli: the 'error-free' system which is inhibited by caffeine and the 'error-prone' system which is inhibited by arsenite. Using spores of Streptomyces cattleya NRRL 8057 and the virulent actinophage VC11 we have shown that a caffeine inhibitable, host mediated UV repair system is active in spores during early development. Some evidence was also found for the presence of an arsenite inhibitable UV repair system. The caffeine inhibitable UV repair system was found to be involved in the induction of genetic instability in S. cattleya. The arsenite system may be implicated in the repair of such events. Genetic instability was also induced by single strand breaks in DNA caused by 32P.     

Unexpected conserved non-coding DNA blocks in mammals

The significance of non-coding DNA is a longstanding riddle in the study of molecular evolution. Using a comparative genomics approach, Dermitzakis and colleagues have recently shown that at least some non-coding sequence, frequently ignored as meaningless noise, might bear the signature of natural selection. If functional, it could mark a turning point in the way we think about the evolution of the genome.     

The stochastic model of non-equilibrium mutagen-induced alterations of DNA: implication to genetic instability in cancer

Genetic alterations such as point mutations, chromosomal rearrangements, modification of DNA methylation and chromosome aberrations accumulate during the lifetime of an organism. They can be caused by intrinsic errors in the DNA replication and repair as well as by external factors such as exposure to mutagenic substances or radiation. The main purpose of the present work is to begin an exploration of the stochastic nature of non-equilibrium DNA alteration caused by events such as tautomeric shifts. This is done by modeling the genetic DNA code chain as a sequence of DNA-bit values ('1' for normal bases and '-1' for abnormal bases). We observe the number of DNA-bit changes resulting from the random point mutation process which, in the model, is being induced by a stochastic Brownian mutagen (BM) as it diffuses through the DNA-bit systems. Using both an analytical and Monte Carlo (MC) simulation techniques, we observe the local and global number of DNA-bit changes. It is found that in 1D, the local DNA-bit density behaves like 1/t, the global total number of the switched (abnormal) DNA-bit increases as t. The probability distribution P(b, 0, t) of b(0, t) is log-normal. It is also found that when the number of mutagens is increased, the number of the total abnormal DNA-bits does not grow linearly with the number of mutagens. All analytic results are in good agreement with the simulation results.