真核细胞的房间的染色体在由环境代理人和内长的新陈代谢的副产品的连续袭击下面。在 DNA 导致的损坏通常导致细胞的事件的串联， DNA 损坏反应。DNA 损坏反应的失败能由减少 DNA 修理的效率和忠实导致恶意的开发。NBS1 蛋白质是在对 D...真核细胞的房间的染色体在由环境代理人和内长的新陈代谢的副产品的连续袭击下面。在 DNA 导致的损坏通常导致细胞的事件的串联， DNA 损坏反应。DNA 损坏反应的失败能由减少 DNA 修理的效率和忠实导致恶意的开发。NBS1 蛋白质是在对 DNA 损坏和 chromosomal 正直的维护的细胞的反应起一个关键作用的 MRE11/RAD50/NBS1 建筑群(MRN ) 的一个部件。在 NBS1 基因的变化为 Nijmegen 破裂症候群(NBS ) 负责，给予增加的倾向到恶意的开发的世袭混乱。从病人们在 DNA 双海滨裂缝的修理指向缺乏的 NBS 孤立的房间的 phenotypic 特征。这里，我们在 DNA 损坏反应考察 NBS1 的角色的当前的知识。强调在 DNA 双海滨修理，察觉到并且发信号的 DNA 损坏的调整，房间周期检查点控制和 telomere 稳定性的维护被放在 NBS1 的角色上。展开更多
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 he...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.展开更多
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-str...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.展开更多
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 po...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.展开更多
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 r...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.展开更多
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 ...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.展开更多
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）...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.展开更多
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 immunosorbe...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 (r<sup>2</sup> = 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.展开更多
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...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.展开更多
基金supported by grants from National Natural Sciences Foundation of China (No.30872237)the National Basic Research Program of China(No.2007CB512900)
文摘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.
基金The authors thank Alexandra Surcel and Carey L Hendrix Lord for helpful comments on this manuscript.The work in our laboratory is supported by grants from the National Science Foundation(IBN-0077832,MCB-9896340,MCB-0092075)the National Institutes of Health(R0 1 GM63871)+3 种基金the US Department of Agriculture(2001-35301-10570 and 2003-35301-13313)Wuxing L was partially supported by the Intercollege Graduate Degree Program in Plant PhysiologyHong M gratefully acknowledges the support of the John Simon Guggenheim Foundationthe National Institutes of Health(F33 GM72245-1).
文摘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.
文摘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.
基金supported by the National Key Research and Development Program of China(2017YFC1001102)National Natural Science Foundation of China(81760507)
文摘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.
文摘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.
基金Supported by Shandong Swine Industry Technology System and Science and Technology Planning Program for Basic Research in Qingdao City(12-1-4-14-jch)
文摘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.
文摘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 (r<sup>2</sup> = 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.
文摘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.
