DNA Double-Strand Breaks,Potential Targets for HBV Integration

Hepatitis B virus(HBV)-induced hepatocellular carcinoma(HCC) is one of the most fre-quently occurring cancers.Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis.More and more researches were designed to find the relationship of the two.In this study,we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks(DSBs),one of the most detrimental DNA damage.An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection,then cells were incubated in patients’ serum with high HBV DNA copies and at the same time,DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector.By using nest PCR,the viral DNA was detected at the sites of the break.It appeared that integra-tion occurred between part of HBV x gene and the I-SceI induced breaks.The results suggested that DSBs,as the DNA damages,may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily.It provided a new site to investi-gate the integration.

Comparison of DNA double-strand breaks induced by ^(16)O^(8+) in deproteinized DNA and intact cells

The yield of DNA double-strand breaks (DSBs) is sure to be influenced by theenvironment around DNA molecule. Inverse pulsed-field gel electrophoresis (PIGE) has beenapplied to compare the sensitivity of B16 cells and their DNA in DSBs induced by 75 MeV/u16O8+ beam. Results show that the percentages of DNA released from the plug(PR) in bothkinds of tile samples increase with the dose and approach a similar quasi-threshold of about81%. A simple new equation was presented to calculate the break level of DNA molecules.Within a certain dose, the relationship between the break level and the dose is linear. Theyield of DSBs in deproteinized DNA was 1.11 DSBs/100 Mbp/Gy, while that in intact cells was0.60DSBs/100Mbp/Gy. It is testified that deproteinized DNA is more sensitive to oxygen ionsirradiation than intact cells.

A New Mechanism of Nonrandom Distribution of DNA Double Strand Breaks

Since 1996, it has been widely accepted that the distribution of DNA double strand breaks (DSBs) induced by ionizing radiation is nonrandom. The explanation to this phenomenon is focused in two parts. One is the ionizing characteristic of the particles and the other is the high-ordered configuration of chromosome in eukaryote~[1,2]. As reported before~[3], we revealed the nonrandom distribution of DSBs when the

Exposure to Long Magnetic Resonance Imaging Thermometry Does Not Cause Significant DNA Double-Strand Breaks on CF-1 Mice

The purpose of the study was to investigate if the high gradient strength and slew rate used for long MRI-thermometry monitoring could cause DNA double-stranded breaks (DSBs). To this end, an enzyme-linked immunosorbent assay (ELISA) was used to quantify γH2AX, a molecular marker for DSBs, in the blood of mice after a 6-hour exposure to magnetic resonance imaging (MRI). Fourteen CF-1 female mice were separated into 4 experimental groups: Untreated negative control, MRI-treated, MRI-Control, and exposed to ionizing radiation positive control. Untreated negative control was used as a baseline for ELISA to quantify γH2AX. MRI-treated consisted of a 6-hour continuous magnetic resonance imaging (MRI) echo planar imaging (EPI) sequence with a slew rate of 192 mT/m/s constituting a significantly longer imaging time than routine clinical imaging. MRI-control mice were maintained under the same conditions outside the MRI scanner for 6-hours. Mice in the irradiation group served as a positive control of DSBs and were exposed to either 2 Gy, 5 Gy or 10 Gy of ionizing radiation. DSBs in the blood lymphocytes from the treatment groups were analyzed using the γH2AX ELISA and compared. Total protein concentration in lysates was determined for each blood sample and averaged 1 ± 0.35 mg/mL. Irradiated positive controls were used to test radiation dose-dependency of the γH2AX ELISA assay where a linear dependency on radiation exposure was observed (r2 = 0.93) between untreated and irradiated samples. Mean and standard error mean of γH2AX formation were calculated and compared between each treatment group. Repeated measures 1-way ANOVA showed statistically significant differences between the means of irradiated controls and both the MRI-control and MRI-treated groups. There was no statistically significant difference between the MRI-treated samples and the MRI-control groups. Our results show that long MRI exposure at a high slew rate did not cause increased levels of γH2AX when compared to control mice, suggesting that no increase in DSBs was caused by the long MR thermometry imaging session. The novelty of this work contradicts other studies that have suggested MRI may cause DSBs;this work suggests an alternative cause of DNA damage.

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.

Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes

DNA damage in oocytes can cause infertility and birth defects. DNA double-strand breaks （DSBs） are highly deleterious and can substantially impair genome integrity. Homologous recombination （HR）-mediated DNA DSB repair plays dominant roles in safeguarding oocyte quantity and quality. However, little is known regarding the key players of the HR repair pathway in oocytes. Here, we identified oocyte-specific gene Ooep as a novel key component of the HR repair pathway in mouse oocytes. OOEP was required for efficient ataxia telangiectasia mutated （ATM） kinase activation and Rad51 recombinase （RAD51） focal accumulation at DNA DSBs. Ooep null oocytes were defective in DNA DSB repair and prone to apoptosis upon exogenous DNA damage insults. Moreover, Ooep null oocytes exhibited delayed meiotic maturation. Therefore, OOEP played roles in preserving oocyte quantity and quality by maintaining genome stability. Ooep expression decreased with the advance of maternal age, suggesting its involvement in maternal aging.

Application of Artificially Induced Double-strand Breaks (DSB) and Triplex-forming Oligonucleotides (TFO) in the Improvement of Gene Targeting Efficiency

Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.

Codon evolution in double-stranded organelle DNA: strong regulation of homonucleotides and their analog alternations

In our previous study, complete single DNA strands which were obtained from nuclei, chloroplasts and plant mitochondria obeyed Chargaff’s second parity rule, although those which were obtained from animal mitochondria deviated from the rule. On the other hand, plant mitochondria obeyed another different rule after their classification. Complete single DNA strand sequences obtained from chloroplasts, plant mitochondria, and animal mitochondria, were divided into the coding and non-coding regions. The non-coding region, which was the complementary coding region on the reverse strand, was incorporated as a coding region in the forward strand. When the nucleotide contents of the coding region or non-coding regions were plotted against the composition of the four nucleotides in the complete single DNA strand, it was determined that chloroplast and plant mitochondrial DNA obeyed Chargaff’s second parity rule in both the coding and non-coding regions. However, animal mitochondrial DNA deviated from this rule. In chloroplast and plant mitochondrial DNA, which obey Chargaff’s second parity rule, the lines of regression for G (purine) and C (pyrimidine) intersected with regression lines for A (purine) and T (pyrimidines), respectively, at around 0.250 in all cases. On the other hand, in animal mitochondrial DNA, which deviates from Chargaff’s second parity rule, only regression lines due to the content of homonucleotides or their analogs in the coding or non-coding region against those in the complete single DNA strand intersected at around 0.250 at the horizontal axis. Conversely, the intersection of the two lines of regression (G and A or C and T) against the contents of heteronucleotides or their analogs shifted from 0.25 in both coding and non-coding regions. Nucleotide alternations in chloroplasts and plant mitochondria are strictly regulated, not only by the proportion of homonucleotides and their analogs, but also by the heteronucleotides and their analogs. They are strictly regulated in animal mitochondria only by the content of homonucleotides and their analogs.

Mechanical properties of double-stranded DNA biofilm with Gaussian distribution

In microcantilever-based label-free biodetection technologies, deflection changes induced by adsorptions of double-stranded DNA （dsDNA） molecules on Au-layer surface are greatly affected by the mechanical, thermal and electrical properties of DNA biofilm. In this paper, the elastic properties of dsDNA biofilm are studied. First, the Parsegian＇s empirical potential based on a mesoscopic liq- uid crystal theory is employed to describe the interaction energy among coarse-grained DNA cylinders. Then, con- sidering a Gaussian distribution of DNA interaxial distance, the thought experiment method is used to derive an analyti- cal expression for Young＇s modulus of DNA biofilm with a stochastic packing pattern for the first time. Results show that Young＇s modulus of DNA biofilm is on the order of 10 MPa. These findings could provide a simple and effective method to evaluate the mechanical properties of soft biofilm on snbstrate.

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18

Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy.

DETECTION OF STRAND BREAKS OF DNA IN HUMAN EARLY CHORIONIC VILLUS CELLS INDUCED BY DIAGNOSTIC ULTRASOUND USING ^(32)P-LABELED ALU HYBRIDIZATION

Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and double-stranded breaks in human DNA. Methods 60 normal pregnant women aged 20-30, who underwent artificial abortion during 6-8 weeks of gestation, were randomly divided into 2 experimental groups: All 30 cases were exposed to diagnostic ultrasound in uterus for 10 minutes, and 24 hours later chorionic villi were extracted; the other 30 cases were taken as the control group. Single-stranded DNA and double-stranded DNA in villus cells in all cases were isolated by the alkaline unwinding combined with hydroxylapatite chromatography, and were quantitatively detected using 32 P-labeled Alu probe for dot-blotting hybridization. Results There was no significant difference in quantity and percentage in single-stranded DNA and double-stranded DNA between 2 groups (P>0.05). 32 P-Alu probe could only hybridize with human DNA, and could detect DNA isolated from as few as 2.5×10 3 chorionic villus cells and 0.45ng DNA in human leukocytes. Conclusion The results suggested that there were no DNA strand damages in human chorionic villus cells when the uterus was exposed to diagnostic ultrasound for 10 minutes. The method,^(32)P-Alu probe for dot-blotting hybridization, was even more specific, sensitive and accurate than conventional approaches.

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

DNA聚合酶θ:易错的多功能DNA末端修复分子

DNA聚合酶θ(DNA polymerase theta,Polθ)是一种广泛存在于动植物中的DNA修复酶。它在选择性末端连接(alternative end-joining,Alt-EJ)途径中发挥着关键作用,常参与DNA双链断裂(DNA double-strand breaks,DSB)损伤修复。在正常生理状态下,Polθ主要调控基因组稳定性。然而,在恶性肿瘤发生时,Polθ表现出异常高表达水平,并参与调控肿瘤细胞的恶性转变过程。研究表明,抑制Polθ活性可导致同源重组(homologous recombination,HR)缺陷的肿瘤细胞发生合成致死(synthetic lethality,SL)。因此,已经开发出多种针对Polθ的小分子抑制剂,可与其他化疗药物联合使用以抑制恶性肿瘤的发展。此外,敲除或抑制Polθ活性还能增加HR修复效率,从而提高外源基因靶向整合效果。本文综述了Polθ及其介导的Alt-EJ修复机制在生物学功能方面的最新研究进展,为靶向Polθ在肿瘤治疗和基因编辑方面的应用提供理论基础。

Effect of prolonging interval time between coronary angiography and percutaneous coronary intervention on X-ray-induced DNA double-strand breaks in blood lymphocytes

Background It is desirable to minimize the risk of adverse radiation effects associated with percutaneous coronary intervention.The aim of this study was to determine the impact of prolonging the interval between coronary angiography and percutaneous coronary intervention on X-ray-induced DNA double-strand breaks in blood lymphocytes using γ-H2AX immunofluorescence microscopy.Methods Blood samples of eight patients were taken before the first exposure to ionizing radiation,10 minutes,20 minutes,30 minutes,1 hour,and 24 hours after the last exposure to determine the γ-H2AX foci repair kinetics.Fifty-eight patients undergoing percutaneous coronary intervention were randomized to an intermittent radiation exposure group and a continuous radiation exposure group.Blood samples were taken before coronary angiography and 15 minutes after the last exposure.By enumerating γ-H2AX foci,the impact of prolonging the interval on DNA double-strand breaks was investigated.Student t-test was used to compare the difference in DNA double-strand breaks between the two groups.Results An increase in foci was found in all patients received percutaneous coronary intervention.The maximum number of γ-H2AX foci was found 10-20 minutes after the end of the last exposure.There was no statistically significant difference between the two groups in γ-H2AX foci at baseline.On average there were （0.79±0.15） γ-H2AX foci induced by interventional X-rays per lymphocyte in the continuous radiation exposure group and （0.66±0.21） in the intermittent radiation exposure group after exposure （P〈0.05）.Conclusions A significant number of γ-H2AX foci develop following the percutaneous coronary intervention procedures.The number of X-ray-induced DNA double-strand breaks may be decreased by prolonging the interval time between coronary angiography and percutaneous coronary intervention to 30 minutes.

川芎嗪通过RAD52调控乳腺癌细胞DNA损伤修复

目的:探究TMP对乳腺癌BT474细胞增殖、细胞周期及其调控蛋白表达与DNA双链断裂修复通路的相关性。方法:CCK8法测定TMP对乳腺癌BT474细胞的增殖抑制情况;流式细胞术测定TMP对细胞周期的影响;单细胞凝胶电泳测定分析TMP对损伤后细胞DSBs累积情况的影响;Isce-I内切酶系统检测TMP对修复通路活性的影响;Western blotting检测DSBs修复通路相关染色体结合蛋白表达水平变化。结果:TMP通过使细胞阻滞在G_(1)期呈浓度依赖性抑制BT474细胞增殖,显著减少体内由Zeocin导致的细胞拖尾DNA含量(P<0.05);TMP显著增加BT474细胞对RAD52、ERCC1、XRCC4以及DNA LigⅣ蛋白募集,减少对KU80蛋白募集,促进了SSA以及NHEJ通路修复活性(P<0.05)。结论:TMP通过阻滞BT474细胞停留在G_(1)期使其发挥增殖抑制作用的机制之一;TMP通过增强损伤缺口对各个通路的关键染色体结合蛋白募集,促进SSA与NHEJ修复通路活性从而减少DNA损伤。

月球辐射环境致DNA链断裂损伤规律的仿真研究

月球表面缺乏磁场和大气的保护,其辐射环境长期处于极端恶劣的水平,对探月航天员的健康构成了严重威胁。针对月表面无屏蔽、航天服屏蔽和月面基地屏蔽条件下的空间辐射环境,运用基于蒙特卡洛方法的Geant4-DNA仿真软件,研究了单细胞核内DNA在上述环境下的链断裂损伤规律。结果表明:质子引起的双链断裂比例低于铁离子;无屏蔽状态下,若爆发太阳粒子事件,DNA结构将在短期内受到严重破坏,链断裂总数可达2×10^(6)个以上,约占细胞核内碱基对总数的0.039%;银河宇宙线环境中,辐射风险主要由长期累计剂量引起,质子凭借更高的通量,拥有了比铁离子更强的DNA破坏效果;但在有屏蔽状态下,DNA结构的损伤状况得到了明显改善,尤其是爆发太阳粒子事件这种极端情况下,最多减少了99.96%的链断裂数。

The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double- Strand Break Repair-Based Insertion Mechanism

Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host＇s DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34 000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double- strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.

重离子辐照诱导DNA双链断裂的剂量率效应

利用不同剂量率的 5 0MeV/u12C6+ 辐照B16黑色素瘤细胞的脱蛋白DNA ,采用脉冲场凝胶电泳技术对DNA双链断裂 (DSB)的诱导和片段的分布进行了研究。结果发现 ,在剂量率分别为3Gy/min和 30Gy/min的情况下 ,DNA片段释放百分比 (PR)都随着剂量的增加而增加 ,并在超过一定剂量之后趋于相似的准阈值 ;3Gy/min辐照诱导DSB的产额为 0 .4 0DSBs/ (10 0Mbp .Gy) ,30Gy/min辐照诱导的DSB产额准确值无法得到 ;30Gy/min辐照诱导DSB的截面为 2 .14μm2 ,是 3Gy/min的 3.1倍。所有结果都表明剂量率越高 ,诱导DSB越有效。另外 ,3Gy/min辐照诱导DSB片段在 -86 0kbp处有一个片段峰 ,而 30Gy/min没有 ,说明剂量率可以影响DSB片段的分布。

基于SYBR Green I的双链DNA定量方法

基于SYBR Green Ⅰ荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA定量方法。将梯度稀释后的基因组DNA及已知浓度的λDNA与等体积的SYBR Green Ⅰ（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2ng/μl,而SYBR Green Ⅰ荧光定量法的检测限可达到0.015ng/μl,并且在0.015-2ng/μl范围内,SYBR Green Ⅰ荧光强度与λDNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。