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.

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.

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.

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.

DSB修复基因hKu70反义RNA真核表达载体构建

目的 构建人DNA双链断裂 (DSB)修复基因hKu70反义RNA真核表达载体pEGFP C1 K ,为以后的hKu70基因功能和毒理学研究提供实验材料。方法 提取人胚肺成纤维细胞 (HLF)总RNA ,逆转录酶 多聚酶链式反应 (RT PCR)扩增hKu70基因cDNA保守序列 ,经与pGEM T载体连接、筛选、克隆、抽提质粒和双酶切后 ,将纯化的hKu70基因cDNA保守序列反向插入绿色荧光蛋白表达载体pEGFP C1中 ,筛选、克隆、抽提质粒 ,从而构建hKu70基因反义RNA真核表达载体pEGFP C1 K。结果 经RT PCR获得 467bp含限制性内切酶位点的DNA片段 ,T载体克隆后经双酶切、测序 ,确定该片段为hKu70基因cDNA ,进而构建反义RNA真核表达载体pEGFP C1 K ,并双酶切 ,测序确证。结论 成功构建hKu70基因反义RNA真核表达载体pEGFP C1 K ,为建立该基因低表达细胞株。

DSB修复蛋白ATM DNA-PKcs与肿瘤放射敏感性关系的研究进展

放射线主要通过导致细胞DNA双链断裂(DSB)而起到杀死肿瘤细胞的作用,但细胞都有不同程度的DSB修复能力,研究证实DSB修复水平与细胞放射敏感性关系密切。在人类细胞内有2种DSB修复途径:一种是以DNA依赖蛋白激酶(DNA-PK)复合物为主的非同源末端连接(NHEJ)修复,另一种是以毛细血管扩张性共济失调症突变蛋白(ATM)为主的同源重组(HR)修复在人体内,NHEJ修复是最主要的修复途径,DNA依赖蛋白激酶催化亚单位(DNA-PKcs)是DNA-PK复合物的主要功能单位。DNA-PKcs的激酶活性是NHEJ修复所必须的,其在DSB修复中起核心作用近年来的研究显示ATM、DNA-PKcs蛋白表达水平与肿瘤放射敏感性有关。该综述将有关ATM、DNA-PKcs的功能、在肿瘤组织中的表达及与肿瘤放射敏感性关系的研究进行简要回顾。

与真核生物DSBs修复有关的NHEJ途径研究进展

DNA双链断裂是真核生物最严重的DNA损伤形式。如果断裂的DNA双链无法及时修复,将可能导致细胞死亡。非同源末端连接途径在真核生物DSBs修复中起重要作用。综述了真核生物NHEJ途径中核心蛋白质Ku、DNA-PKcs、DNA连接酶IV、XRCC4、ARTEMIS和XIF等因子的结构和功能,并简要介绍了NHEJ修复途径的分子机制,其中涉及到DSBs位点蛋白复合体组装的两种模型。

BRCA1与DSB修复路径取向

乳腺癌易感基因1(BRCA1)是一个肿瘤抑制基因.BRCA1参与DNA末端切除、细胞周期调控以及染色体修饰等来维护基因组的稳定性.有研究表明,它能够促进正确的DNA双链断裂(DSBs)修复,如同源重组修复(HDR)和经典的非同源末端连接(C-NHEJ);而抑制错误性的DSB修复,如单链退火修复(SSA)和非经典的末端连接(A-EJ);其机制是通过与某些DNA修复相关蛋白质的相互作用来引导DSB修复.目前,BRCA1在DSB修复通路中的作用机制尚未完全明确,仍有待进一步的研究.本文主要阐述BRCA1在DSB各修复通路中是如何发挥其引导作用的.

基于DSBs修复蛋白表达构建预测食管鳞癌放化疗预后的列线图模型

目的探讨DNA双链断裂(DSBs)修复蛋白ATM、DNA-PKcs及P16表达水平与根治性放化疗食管鳞癌患者远期生存的关系,并建立列线图预测模型。方法收集2012-2017年收治的283例根治性放化疗食管鳞癌病例,按2:1随机分为建模组和验证组。免疫组化检测DSBs修复蛋白在肿瘤中的表达,Kaplan-Meier法计算生存率,Log-rank法进行单因素分析,Cox模型进行多因素分析,R软件构建列线图模型并进行内外部验证。结果建模组患者中位随访34个月,3、5年总生存率为35.1%和20.0%。多因素分析显示,ATM、DNA-PKcs或P16的表达与否,以及T、N分期均是患者OS的预后因素。建立列线图模型,预测建模组患者3、5年OS的一致性指数C-index为0.724,显著高于第七版AJCC临床分期的0.533(P <0.001)。对验证组患者进行验证,本模型和AJCC分期的C-index分别为0.792和0.550(P <0.001),均提示本模型比AJCC分期准确性更高。结论基于DSBs修复蛋白表达建立的列线图模型能较好地预测根治性放化疗食管鳞癌患者的远期生存,对指导个体化治疗有一定的临床意义。

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。

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。

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.

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。

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。

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.