DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells

DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.Subject terms: Double-strand DNA breaks, Cell signalling     

Functional insights gained from structural analyses of DNA duplexes that contain UV-damaged photoproducts

Ultraviolet photolesions endow DNA with distinct structural and dynamic properties. Biophysical studies of photoproduct-containing DNA have shown that these lesions affect the mutagenic properties of DNA and damage recognition by DNA repair systems. Recently obtained high-resolution cocrystal structures of damaged DNA bound to either DNA polymerase or DNA repair enzymes have enriched our understanding of the mechanisms by which DNA lesions are bypassed or recognized by DNA metabolizing proteins. Here, we summarize the results of these structural studies and discuss their implications for DNA metabolism.     

High-density DNA alignment on an Au(111) surface starting from folded DNA

High-density uniform DNA alignment on a metal substrate is essential for creating sensitive DNA devices. We develop a self-sensing DNA alignment process starting from folded DNA to achieve high-density, uniform DNA alignment on an Au(111) surface. We demonstrate that folded DNA plays a critical role in avoiding DNA aggregation and distributing the DNA uniformly on an Au(111) surface at the greatest density and quality ever attained. We also verify that the distributed, folded DNA can be stimulated to align only when the appropriate buffer flow is applied. This selective self-sensing DNA alignment on an Au surface will be a key technology for creating dynamic DNA sensors and switches.     

Thermodynamics of DNA condensation caused by mn2+ binding

Interaction between Mn2+ ion and the two forms of DNA duplex (supercoiled and linearized pUC119 DNA) in solution has been examined by isothermal titration calorimetry. Although DNA condensation reaction heat was observed at 323 K, this was not the case at 298 K. DNA condensation was entropically driven and supercoiled DNA was found to be more susceptive. The enthalpy of DNA condensation is estimated 0.42 kJ/mol for both DNA forms. Conversely, the entropy of DNA condensation was 0.13 kJ/mol K for supercoiled DNA, and 0.12 kJ/mol K for linearized DNA. The difference of entropy is attributable to their DNA conformation.     

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.     

三螺旋脱氧核糖核酸的研究进展

阐述了三螺旋DNA的发展和最新动态,并从3个方面展开评述：(1)三螺旋DNA稳定性的研究。三螺旋DNA的稳定性不仅取决于寡聚核苷酸的内在结构,还受外界环境如溶液的pH值、阳离子的种类和价态、DNA分子嵌入试剂和共聚物等的影响;(2)三螺旋DNA的应用。三螺旋DNA的形成为基因操纵和基因疗法提供了新方法,它在抑制DNA转录、复制、基因定点诱变、定点切割和诱导基因重组等方面有重要的应用前景;(3)三螺旋DNA的检测方法,包括紫外-可见吸收光谱法、荧光分析法、原子力显微术和顺磁共振谱等。     

Manipulation of globular DNA molecules for sizing and separation

Handling large DNA molecules, such as chromosomal DNA, has become necessary due to recent developments in genome science. However, large DNA molecules are fragile and easily broken by shear stress accompanying flow in solution. This fragility causes difficulties in the preparation and handling of large DNA molecules. This study demonstrates the transition of DNA from a coiled to a globular form, which is highly condensed. This state suppresses DNA fragmentation due to shear stress in solution. The transition enables large DNA molecules to undergo mechanical manipulation. We confirmed that the fluorescence intensity of stained globular DNA increases with increasing length, suggesting that the resistance of globular DNA to shear stress is the factor that allows analysis of large DNA by flow cytometry.     

Effects of photo-activated bleomycin on deoxyribonuclease I, exonuclease III and deoxyribonucleic acid polymerase I reactions

The activity of bleomycin to break the strand of deoxyribonucleic acid (DNA) in the presence of 2-hydroxy-1-ethanethiol (2-mercaptoethanol) was enhanced by ultraviolet (UV) irradiation. Photo-activated bleomycin stimulated the action of deoxyribonuclease I (DNase I) to degrade DNA and the DNA synthesis by DNA polymerase I with DNase I. On the other hand, although UV-irradiated bleomycin scarcely broke the DNA strand in the presence of 1,2-benzenediol (catechol), it stimulated the action of DNase I to degrade DNA in the presence of catechol. In accordance with the inhibition by catechol, when DNA treated with UV-irradiated bleomycin in the presence of catechol was employed as a primer for the DNA synthesis, the incorporation of precursor into the acid-insoluble fraction by DNA polymerase I with exonuclease III was reduced to about one-half of the incorporation into DNA treated with unirradiated bleomycin. These findings suggest that the ability of bleomycin to bind to double-helical DNA forming regions sensitive to DNase I was increased by an appropriate dose of UV irradiation and that catechol inhibited the activity of the UV-irradiated bleomycin to break the DNA strand rather than to bind to DNA.     

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O⁶-methylguanine (O⁶-MeG), O⁶-carboxymethylguanine (O⁶-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

DNA分子结构的多态性研究 (Ⅱ) 吖啶橙凝聚体变化诱导的质粒DNA大分子构象的研究

利用吸收光谱和荧光光谱方法,研究了吖啶橙（ＡＯ）与质粒ＤＮＡ水溶液、以及含胶束介质的吖啶橙与质粒ＤＮＡ溶液体系的相互结合作用及减色效应。结果表明：吖啶橙对质粒ＤＮＡ的吸收光谱有减色效应;含十二烷基硫酸钠（ＳＤＳ）的ＡＯ水溶液体系中,随着ＳＤＳ浓度的增加,其光谱结果表现为由凝聚态向单体的转化。而在含十二烷基硫酸钠（ＳＤＳ）的ＡＯ与质粒ＤＮＡ溶液体系中,吖啶橙凝聚态随ＳＤＳ浓度的增加,对ＡＯ与质粒ＤＮＡ相互结合产生协同的减色效应,使质粒ＤＮＡ空间结构发生缩拢。进一步采用电泳法研究了ＡＯ凝聚态可能对质粒ＤＮＡ构象的影响,结果表明：在ＡＯ与质粒ＤＮＡ溶液体系中,ＡＯ浓度的增加对质粒ＤＮＡ构象未产生影响;而在含有ＳＤＳ的ＡＯ与质粒ＤＮＡ的溶液体系中,由于ＳＤＳ对ＡＯ凝聚态的解聚作用,以及ＳＤＳ对质粒ＤＮＡ减色效应的协同作用,使得质粒ＤＮＡ的构象发生变化,诱导质粒ＤＮＡ形成超螺旋构象     

Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe.

A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe. The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA). The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA. The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied. The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction.     

电化学DNA生物传感器*

对特异DNA序列的检测在基因相关疾病的诊断、军事反恐和环境监测等方面均具有非常重要的意义,DNA传感器的研究就是为了满足对特异DNA序列的快速、便捷、高灵敏度和高选择性检测的需要。近年来涌现出了多种传感策略,根据检测方法的不同可以大致分为光学传感器、电化学传感器、声学传感器等。由于电化学检测方法本身所具有的灵敏、快速、低成本和低能耗等特点,电化学DNA传感器已成为一个非常活跃的研究领域并在近几年中得到了快速发展。本文概括了近年来在DNA传感器的重要分支——电化学DNA传感器领域内的一些重要进展,主要包括DNA探针在传感界面上的固定方法和各种电化学DNA杂交信号的检测方法。     

Complexation of DNA with cationic surfactants as studied by small-angle X-ray scattering

The phase behaviors of the complex formed by didodecyldimethylammonium bromide(DDAB)and cetyltrimethylammonium bromide(CTAB)interacting with three different types of DNAs,salmon testes DNA(~2000 bp),21-bp double-stranded oligonucleotides(oligo-ds DNA),and 21-nt single-stranded oligonucleotides(oligo-ss DNA)were studied by synchrotron small-angle X-ray scattering.It was found that the DNA length and flexibility,together with the positive/negative charge ratio,determined the final structure.At higher charge ratios,the DNA length exhibited negligible effect.Both oligo-ds DNA and salmon DNA formed inverted hexagonal packing of cylinders with CTAB,as well as bilayered lamella with DDAB.However,at lower charge ratios,oligo-ds DNA formed a distorted hexagonal phase with CTAB and a new structure with DDAB,which was different from the behaviors of salmon DNA.The flexible oligo-ss DNA formed rich structures that were subject to environmental disturbance.Kinetic study also indicated that the structures of the complex formed by oligo-ss DNA took much longer to mature than the structures formed by oligo-ds DNA.We attributed this result to the conformational adjustment of oligo-ss DNA in the complex.     

Electrochemical and Spectroscopic Study of the Interaction of Indirubin with DNA

The interaction of indirubin with DNA was studied by differential pulse voltammetry (DPV) and linear sweep voltammetry (LSV) at the bare or DNA‐modified electrode and UV‐vis or IR spectra. As a result of intercalating of this drug into the double helical structure of DNA, the DPV of indirubin shows that peak potentials shift and peak currents decrease with the addition of DNA. UV‐vis spectra exhibits that the absorption intensity of indirubin at 538.7 nm^?1 decreases, which indicates that the anticancer herbal drug indirubin binds DNA. In addition, IR‐spectra of DNA and DNA‐indirubin adduct imply indirubin interacts with the phosphate groups of DNA by hydrogen bond or electrostatic interaction. Under our experiment conditions, the decrease of peak current is proportional to DNA concentration, which can be applied to estimate DNA concentration. The results indicate that the herbal drug indirubin can interfere with the DNA by intercalating into the double helix of DNA and interacting with the phosphate groups of DNA.     

DNA and RNA sensor

The development of DNA sensors attracts recentresearch attention directed at gene analysis, such asdetection of genetic disorders, tissue matching andforensic applications[1,2]. In general, sensors based on the principle differ-ent from that of the tr…     

酸性条件下的DNA构象变化加速DNA变性解链

调控DNA的变性解链过程是DNA扩增与检测的关键步骤。对于传统的热循环DNA扩增策略,由于变温过程中热量分布不均一以及变温速度慢等不利因素,会直接影响DNA变性解链过程,从而降低DNA检测放大的效果、延长检测的时长。因此,探索快速、高效的调控DNA变性解链的方法具有重要的研究意义。本文发展了以胞嘧啶在酸性条件下的质子化反应为基础,通过改变溶液的pH值,来诱导DNA构象在Watson-Crick（WC）碱基对与Hoogsteen（HG）碱基对之间的分子构象转换,从而实现精准、快速、高效的DNA变性解链调控目标。结果表明,相较于传统的温控方法,pH调控方法能显著提高DNA变性的速率约6倍以上。本文发现pH调控方法通过降低双链DNA的反应焓约160 kJ/mol,从而提高双链DNA变性速率和效率。该方法具有用于DNA信号放大与检测等相关应用的潜力。     

New acridone derivatives for the electrochemical DNA-hybridisation labelling

In the field of DNA sensing, DNA hybridisation detection is generally performed by fluorescence microscopy. However, fluorescence instrumentation is difficult to miniaturise in order to produce fully integrated DNA chips. In this context, electrochemical detection of DNA hybridisation may avoid this limitation. Therefore, the use of DNA intercalators is particularly attractive due to their selectivity toward DNA double strand enabling DNA labelling without target chemical modification and, for most of them, to their electroactivity. We have synthesized a pyridoacridone derivative dedicated to DNA hybridisation electrochemical-sensing which presents good electrochemical reversibility, electroactivity at mild potentials and specificity toward DNA double strand. The electrochemical behaviour of this molecule has been assessed using cyclic voltammetry (CV). DNA/intercalator interactions were studied by differential pulse voltammetry (DPV) before application to hybridisation detection onto DNA sensors based on polypyrrole modified electrodes.     

丝组二肽对DNA的切割作用的研究

近20年来,人工核酸切割试剂的研究一直是生物化学中最为活跃的前沿领域之一,研究人工核酸切割试剂的主要目的是合成定点切割试剂,后者是一种重要的分子生物学工具,在疾病的基团治疗、反义PCR技术等领域中有着重要的应用价值。此外,人工核酸切割试剂还可以在足迹技术和核酸高级结构的研究中用作高分辨率的化学探针。