Duplications between direct repeats stabilized by DNA secondary structure occur preferentially in the leading strand during DNA replication

To ascertain a leading or lagging strand preference for duplication mutations, several short DNA sequences, i.e. mutation inserts, were designed that should demonstrate an asymmetric propensity for duplication mutations in the two complementary DNA strands during replication. The design of the mutation insert involved a 7-bp quasi inverted repeat that forms a remarkably stable hairpin in one DNA strand, but not the other. The inverted repeat is asymmetrically placed between flanking direct repeats. This sequence was cloned into a modified chloramphenicol acetyltransferase (CAT) gene containing a −1 frameshift mutation. Duplication of the mutation insert restores the reading frame of the CAT gene resulting in a chloramphenicol resistant phenotype. The mutation insert showed greater than a 200-fold preference for duplication mutations during leading strand, compared with lagging strand, replication. This result suggests that misalignment stabilized by DNA secondary structure, leading to duplication between direct repeats, occurred preferentially during leading strand synthesis.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.     

Topoisomerase I Alone Is Sufficient to Produce Short DNA Deletions and Can Also Reverse Nicks at Ribonucleotide Sites

Ribonucleotide monophosphates (rNMPs) are among the most frequent form of DNA aberration, as high ratios of ribonucleotide triphosphate:deoxyribonucleotide triphosphate pools result in approximately two misincorporated rNMPs/kb of DNA. The main pathway for the removal of rNMPs is by RNase H2. However, in a RNase H2 knock-out yeast strain, a topoisomerase I (Top1)-dependent mutator effect develops with accumulation of short deletions within tandem repeats. Proposed models for these deletions implicated processing of Top1-generated nicks at rNMP sites and/or sequential Top1 binding, but experimental support has been lacking thus far. Here, we investigated the biochemical mechanism of the Top1-induced short deletions at the rNMP sites by generating nicked DNA substrates bearing 2′,3′-cyclic phosphates at the nick sites, mimicking the Top1-induced nicks. We demonstrate that a second Top1 cleavage complex adjacent to the nick and subsequent faulty Top1 religation led to the short deletions. Moreover, when acting on the nicked DNA substrates containing 2′,3′-cyclic phosphates, Top1 generated not only the short deletion, but also a full-length religated DNA product. A catalytically inactive Top1 mutant (Top1-Y723F) also induced the full-length products, indicating that Top1 binding independent of its enzymatic activity promotes the sealing of DNA backbones via nucleophilic attacks by the 5′-hydroxyl on the 2′,3′-cyclic phosphate. The resealed DNA would allow renewed attempt for repair by the error-free RNase H2-dependent pathway in vivo. Our results provide direct evidence for the generation of short deletions by sequential Top1 cleavage events and for the promotion of nick religation at rNMP sites by Top1.     

i-Motif DNA: Structure,stability and targeting with ligands

i-Motifs are four-stranded DNA secondary structures which can form in sequences rich in cytosine. Stabilised by acidic conditions, they are comprised of two parallel-stranded DNA duplexes held together in an antiparallel orientation by intercalated, cytosine–cytosine⁺ base pairs. By virtue of their pH dependent folding, i-motif forming DNA sequences have been used extensively as pH switches for applications in nanotechnology. Initially, i-motifs were thought to be unstable at physiological pH, which precluded substantial biological investigation. However, recent advances have shown that this is not always the case and that i-motif stability is highly dependent on factors such as sequence and environmental conditions. In this review, we discuss some of the different i-motif structures investigated to date and the factors which affect their topology, stability and dynamics. Ligands which can interact with these structures are necessary to aid investigations into the potential biological functions of i-motif DNA and herein we review the existing i-motif ligands and give our perspective on the associated challenges with targeting this structure.     

三核苷酸重复序列失稳定机制:重复序列形成non-B二级结构的必要性和细胞反式作用因子的作用

与三核苷酸重复序列CAG.CTG、CGG·CCG和GAA·TTC扩增和缺失有关的分子机制尚不能得到清楚的阐释.体外研究表明,上述疾病相关的重复序列可以在体外形成non-B二级结构,并介导重复序列扩增.然而,迄今为止,类似的观察尚未在体内研究过程中得以实现.利用模型生物大肠杆菌和酵母等进行的有关研究并不能模拟三核苷酸重复序列的扩增,这暗示三核苷酸重复序列的体内扩增可能与重复序列形成non-B二级结构关联性并不大.尽管理论上较长的三核苷酸重复序列可以在复制和后复制过程中较易形成non-B DNA二级结构,但这样的二级结构倾向于导致重复序列出现"脆性",而不是扩增.事实上,患者所具有的三核苷酸重复序列扩增并非一定需要通过non-B二级结构的介导,这些重复序列的扩增是可以通过一种RNA转录诱导的局部DNA重复序列的复制和其后的DNA重排得以发生.     

Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair

Expansion of triplex-forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN, there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSbeta and endonuclease activity of MutLalpha. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.     

The role of the Fanconi anemia network in the response to DNA replication stress

Fanconi anemia is a genetically heterogeneous disorder associated with chromosome instability and a highly elevated risk for developing cancer. The mutated genes encode proteins involved in the cellular response to DNA replication stress. Fanconi anemia proteins are extensively connected with DNA caretaker proteins, and appear to function as a hub for the coordination of DNA repair with DNA replication and cell cycle progression. At a molecular level, however, the raison d’être of Fanconi anemia proteins still remains largely elusive. The thirteen Fanconi anemia proteins identified to date have not been embraced into a single and defined biological process. To help put the Fanconi anemia puzzle into perspective, we begin this review with a summary of the strategies employed by prokaryotes and eukaryotes to tolerate obstacles to the progression of replication forks. We then summarize what we know about Fanconi anemia with an emphasis on biochemical aspects, and discuss how the Fanconi anemia network, a late acquisition in evolution, may function to permit the faithful and complete duplication of our very large vertebrate chromosomes.     

Condensed Representations of Protein Secondary Structure Sequences and Their Application

Abstract

In this paper, we propose a nongraphical representation for protein secondary structures. By counting the frequency of occurrence of all possible four-tuples (i.e., four-letter words) of a protein secondary structure sequence, we construct a set of 3 × 3 matrices for the corresponding protein secondary structure sequence. Furthermore, the leading eigenvalues of these matrices are computed and considered as invariants for the protein secondary structure sequences. To illustrate the utility of our approach, we apply it to a set of real data to distinguish protein structural classes. The result indicates that it can be used to complement the classification of protein secondary structures.     

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

The removal of initiating primers from the 5′-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.     

Role of deoxyridine incorporation and DNA repair in the expression of human chromosomal fragile sites

This paper is a discussion of the possible roles of deoxyuridine incorporation into DNA and DNA-repair processes in the expression of the folate-sensitive, common chromosomal fragile sites. Expression of aberrations at these sites increases under conditions expected to increase deoxyuridine incorporation into the chromosome. It is likely that this abnormal base is removed by an excision-repair process that results in transient chromosome breaks; these breaks are seen as chromosome aberrations if repair is not completed before metaphase. Analogous events may account for other types of chromosome aberrations including the so-called “spontaneous” aberrations, the rare folate-sensitive fragile sites, and fragile sites induced by other means.     

RNA二级结构在微生物系统发育分析上的应用

RNA二级结构用于微生物分类和系统发育分析在近年来逐渐受到注意并发展起来。文章就此方面的发展做了简要综述,其中对在系统发育分析上的应用部分作了较重点介绍。     

16S rRNA二级结构的研究进展及其在系统分类中的应用

16S rRNA作为研究系统进化的生物大分子极受重视,是系统分类中必不可少的一个指标。然而,在进行系统发育分析时通常的序列联配方法有明显的局限性,即无法摆脱一级结构的束缚。20世纪80年代以来,一些研究者把目光投向了16S rRNA二级结构的分析研究,并且已经取得了相当不错的进展。研究表明,16S rRNA二级结构可能作为一个新的分类指标,成为系统分类的一个重要补充。     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

Fuzzy Kernel Clustering of RNA Secondary Structure Ensemble Using a Novel Similarity Metric

Abstract

Measuring the (dis)similarity between RNA secondary structures is critical for the study of RNA secondary structures and has implications to RNA functional characterization. Although a number of methods have been developed for comparing RNA structural similarities, their applications have been limited by the complexity of the required computation. In this paper, we present a novel method for comparing the similarity of RNA secondary structures generated from the same RNA sequence, i.e., a secondary structure ensemble, using a matrix representation of the RNA structures. Relevant features of the RNA secondary structures can be easily extracted through singular value decomposition (SVD) of the representing matrices. We have mapped the feature vectors of the singular values to a kernel space, where (dis)similarities among the mapped feature vectors become more evident, making clustering of RNA secondary structures easier to handle. The pair-wise comparison of RNA structures is achieved through computing the distance between the singular value vectors in the kernel space. We have applied a fuzzy kernel clustering method, using this similarity metric, to cluster the RNA secondary structure ensembles. Our application results suggest that our fuzzy kernel clustering method is highly promising for classifications of RNA structure ensembles, because of its low computational complexity and high clustering accuracy.     

DNA微阵列技术在阿尔茨海默病及其防治药物研究中的应用

在生物技术飞速发展的今天,DNA微阵列已成为功能基因组时代大规模、高通量乃至全基因组表达和功能研究的有力工具。阿尔茨海默病,因其发病机制复杂,迄今尚无定论,因此在临床上也缺乏有效的防治药物。简要综述DNA微阵列技术应用于阿尔茨海默病发病机制、早期诊断及防治药物等方面的研究进展。