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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Determination of DNA single-strand breaks by low-energy heavy ion and analysis of dose-effect curves

Calf thymus DNA was exposed to low-energy heavy ions (N+) and 60Co-γ-rays, and the dose-effect on DNA single-strand breaks (SSB) has been investigated. The results indicate that the dose-effect curve by N+ irradiation is different from that of conventional ionizing radiation. While the curve from γ-irradiation follows exponential type, the effect curve produced by N+ ion is of 'saddle type'. The yield of DNASSB per dose unit per DNA unit remained at a certain level under different doses of γ-rays. In contrast, the DNASSB at low dosage region of N+ showed an obvious peak before it decreased rapidly to a lower level.

A NEW METHOD UTILIZED TO DETECT DNA SINGLE STRAND BREAK(NICK-TRANSLATION)

It is often needed to detect the DNA single strand break in biological studies. The four most frequently used methods are alkaline elution, alkaline sucrose gradient sedimentation, hydroxyapatite chromatography and nucleoid sedimentation. All these are not perfect because of a number of disadvantages, such as

Mutual inhibition between miR-34a and SIRT1 contributes to regulation of DNA double-strand break repair

DNA double-strand breaks are repaired through either non-homologous end joining(NHEJ) or homologous recombination repair(HRR) pathway.The well-characterized regulatory mechanisms of double-strand break repair(DSBR) are mainly found at the level of complicated repair protein interactions and modifications.Regulation of DSBR at the transcriptional level was also reported.In this study,we found that DSBR can be regulated by miR-34a at the post-transcriptional level.Specifically,miR-34a,which can be activated by DNA damages,represses DSBR activities by impairing both NHEJ and HRR pathways in cultured cells.The repression is mainly through targeting the critical DSBR promoting factor SIRT1,as ectopically expressed SIRT1 without 3'-UTR can rescue the inhibitory roles of miR-34a on DSBR.Further studies demonstrate that SIRT1 conversely represses miR-34a expression.Taken together,our data show that miR-34a is a new repressor of DSBR and the mutual inhibition between miR-34a and SIRT1 may contribute to regulation of DNA damage repair.

Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in <i>S. cerevisiae</i>

Several modes of eukaryotic of DNA double strand break repair (DSBR) depend on synapsis of complementary DNA. The Rad51 ATPase, the S. cerevisiae homolog of E. coli RecA, plays a key role in this process by catalyzing homology searching and strand exchange between an invading DNA strand and a repair template (e.g. sister chromatid or homologous chromosome). Synthesis dependent strand annealing (SDSA), a mode of DSBR, requires Rad51. Another repair enzyme, the Rad1-Rad10 endonuclease, acts in the final stages of SDSA, hydrolyzing 3￠ overhanging single-stranded DNA. Here we show in vivo by fluo-rescence microscopy that the ATP binding function of yeast Rad51 is required to recruit Rad10 SDSA sites indicating that Rad51 pre-synaptic filament formation must occur prior to the recruitment of Rad1-Rad10. Our data also show that Rad51 ATPase activity, an important step in Rad51 filament disassembly, is not absolutely required in order to recruit Rad1- Rad10 to DSB sites.

“藕断丝连”的CRISPR/Cas:基因编辑中靶点滞留的作用与挑战

成簇规律间隔短回文重复(clustered regulation interspaced short palindromic repeats,CRISPR)和CRISPR相关蛋白质(CRISPR-associated protein,Cas)系统被广泛应用于基因组编辑、转录调控以及细胞实时成像等,并已在农业、工业和医学等领域展示出巨大的应用潜力。该技术的应用取决于CRISPR/Cas的五大属性:靶向、解旋、切割、滞留和旁切。本综述将主要以化脓链球菌(Streptococcus pyogenes)的CRISPR/Cas9为例,聚焦于CRISPR/Cas的滞留属性,梳理相关进展,讨论其在基因编辑技术开发中的应用与挑战。

精细胞中促进组蛋白-鱼精蛋白替换的分子机制研究进展

在精子形成过程中,圆形精子细胞核内染色质中的组蛋白逐渐被鱼精蛋白替代,使染色质凝聚得更为紧凑,奠定成熟精子头部特异形态的结构基础。若组蛋白-鱼精蛋白替换缺陷,将导致精子核凝聚失败,表现出精子形态异常和活力降低,受精困难。研究显示,组蛋白翻译后修饰、瞬时DNA链断裂、DNA链断裂修复和染色质重塑复合体识别等一系列变化都会促进组蛋白-鱼精蛋白的替换,本文从以上几个方面综述了目前对于促进组蛋白-鱼精蛋白替换分子机制的研究进展,以期为雄性不育症的诊断和治疗提供理论基础。

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。

精准序列替换基因组编辑技术研究进展

利用基因组编辑技术可以对微生物、动植物和人类细胞系基因组进行精准的序列替换,加速生物育种进程和遗传性疾病治疗,从而在农业生产和医疗上取得突破。基因组序列替换策略主要分为2种:第1种依赖DNA双链断裂,包括将CRISPR-Cas分别与同源重组、单链退火、微同源末端连接等DNA修复途径相结合,或由位点特异性重组系统介导,实现精准的序列替换;第2种依赖DNA单链断裂,主要包括引导编辑、碱基编辑器等技术。本研究综述了不同精准序列替换策略和技术及相关研究进展,理清各策略和技术的优缺点,有助于根据基因组编辑的目的,选择适合的技术和方法实现精准高效的序列替换。

MRE11-RAD50-NBS1复合物的功能与人类疾病的研究进展

生物体的DNA常遭受着来自体外和体内各种因素的攻击,其中DNA双链断裂(DSB)是严重的一种DNA损伤方式。为了保证遗传信息的稳定性,生物体自身存在应对DNA损伤的修复机制。同源重组修复是精确的修复DSB的方式,MRE11-RAD50-NBS1(MRN)复合物是参与同源重组修复的关键蛋白,在不同物种之间存在保守性。从前关于MRN复合物的功能研究主要来源于酿酒酵母和线虫等低等生物,近些年来对哺乳动物MRN复合物的研究提示MRN复合物在高等动物DNA损伤修复中存在功能。本文综述了MRN复合物的组成和结构及其在DNA损伤同源重组修复中的功能,同时也介绍了MRN复合物异常所带来的人类疾病共济失调性毛细血管扩张综合征类似病症、奈梅亨断裂综合征和奈梅亨断裂综合征类似病症,并对这3类DNA损伤修复缺陷疾病的临床表型和相关小鼠模型研究进行了总结。

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。