Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different ch...Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.展开更多
CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells(Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014).The most widely us...CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells(Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014).The most widely used customized CRISPR/Cas9(Sp Cas9)is derived from Streptococcus pyogenes(Cong et al.,2013).展开更多
DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end ...DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.展开更多
Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the effici...Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.展开更多
Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome ...Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.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
The Szent-Gyorgyi Prize for Progress in Cancer Research is a prestigious scientific award established by the National Foundation for Cancer Research(NFCR)—a leading cancer research charitable organization in the Unit...The Szent-Gyorgyi Prize for Progress in Cancer Research is a prestigious scientific award established by the National Foundation for Cancer Research(NFCR)—a leading cancer research charitable organization in the United States that is committed to supporting scientific research and public education relating to the prevention,early diagnosis,better treatments,and ultimately,a cure for cancer.Each year,the Szent-Gyorgyi Prize honors an outstanding researcher,nominated by colleagues or peers,who has contributed outstanding,significant research to the fight against cancer,and whose accomplishments have helped improve treatment options for cancer patients.The Prize also promotes public awareness of the importance of basic cancer research and encourages the sustained investment needed to accelerate the translation of these research discoveries into new cancer treatments.This report highlights the pioneering work led by the 2015 Prize winner,Dr.Frederick Alt.Dr.Alt's work in the area of cancer genetics over four decades has helped to shape the very roots of modern cancer research.His work continues to profoundly impact the approaches that doctors around the globe use to diagnose and treat cancer.In particular,his seminal discoveries of gene amplification and his pioneering work on molecular mechanisms of DNA damage repair have helped to usher in the era of genetically targeted therapy and personalized medicine.展开更多
Over the past few decades, major strides have advanced the techniques for early detection and treatment of cancer. However, metastatic tumor growth still accounts for the majority of cancer-related deaths worldwide. I...Over the past few decades, major strides have advanced the techniques for early detection and treatment of cancer. However, metastatic tumor growth still accounts for the majority of cancer-related deaths worldwide. In fact, breast cancers are notorious for relapsing years or decades after the initial clinical treatment, and this relapse can vary according to the type of breast cancer. In estrogen receptor-positive breast cancers, late tumor relapses frequently occur whereas relapses in estrogen receptor-negative cancers or triple negative tumors arise early resulting in a higher mortality risk. One of the main causes of metastasis is tumor dormancy in which cancer cells remain concealed, asymptomatic, and untraceable over a prolonged period of time. Under certain conditions, dormant cells can re-enter into the cell cycle and resume proliferation leading to recurrence. However, the molecular and cellular regulators underlying this transition remain poorly understood. To date, three mechanisms have been identified to trigger tumor dormancy including cellular, angiogenic, and immunologic dormancies. In addition, recent studies have suggested that DNA repair mechanisms may contribute to the survival of dormant cancer cells. In this article, we summarize the recent experimental and clinical evidence governing cancer dormancy. In addition, we will discuss the role of DNA repair mechanisms in promoting the survival of dormant cells. This information provides mechanistic insight to explain why recurrence occurs, and strategies that may enhance therapeutic approaches to prevent disease recurrence.展开更多
It is proposed that mismatch repair (MMR) mediates the cytotoxic effects of DNA damaging agents by exerting a futile repair pathway which leads to double strand breaks (DSBs). Previous reports indicate that the sensit...It is proposed that mismatch repair (MMR) mediates the cytotoxic effects of DNA damaging agents by exerting a futile repair pathway which leads to double strand breaks (DSBs). Previous reports indicate that the sensitivity of cells defective in homologous recombination (HR) to DNA alkylation is reduced by defects in MMR genes. We have assessed the contribution of different MMR genes to the processing of alkylation damage in vivo. We have directly visualized recombination complexes formed upon DNA damage using fluorescent protein (FP) fusions. We find that msh6 mutants are more resistant than wild type cells to MNNG, and that an msh6 mutation rescues the sensitivity of rad52 strains more efficiently than an msh3 mutation. Analysis of RAD52-GFP tagged strains indicate that MNNG increases repair foci formation, and that the inactivation of the MHS2 and MSH6 genes but not the MSH3 gene result in a reduction of the number of foci formed. In addition, in the absence of HR, NHEJ could process the MNNG-induced DSBs as indicated by the formation of NHEJ-GFP tagged foci. These data suggest that processing of the alkylation damage by MMR, mainly by MSH2-MSH6, is required for recruitment of recombination proteins to the damage site for repair.展开更多
Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consist...Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.展开更多
DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damag...DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks(DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination(HR) and non-homologous end joining(NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair.Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes(DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.展开更多
Genome stability can be threatened by both endogenous and exogenous agents.Organisms have evolved numerous mechanisms to repair DNA damage,including homologous recombination(HR)and non-homologous end joining(NHEJ).Amo...Genome stability can be threatened by both endogenous and exogenous agents.Organisms have evolved numerous mechanisms to repair DNA damage,including homologous recombination(HR)and non-homologous end joining(NHEJ).Among the factors associated with DNA repair,the MRE11-RAD50-NBS1(MRN)complex(MRE11-RAD50-XRS2 in Saccharomyces cerevisiae)plays important roles not only in DNA damage recognition and signaling but also in subsequent HR or NHEJ repair.Upon detecting DNA damage,the MRN complex activates signaling molecules,such as the protein kinase ataxia-telangiectasia mutated(ATM),to trigger a broad DNA damage response,including cell cycle arrest.The nuclease activity of the MRN complex is responsible for DNA end resection,which guides DNA repair to HR in the presence of sister chromatids.The MRN complex is also involved in NHEJ,and has a species-specific role in hairpin repair.This review focuses on the structure of the MRN complex and its function in DNA damage repair.展开更多
Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break(DSB)signaling.P53-binding protein 1(53BP1)plays a critical role in coordinating the DSB repair pathway c...Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break(DSB)signaling.P53-binding protein 1(53BP1)plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining(NHEJ)-mediated DSB repair pathway that rejoins DSB ends.New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination(HR)signaling.This review focuses on the up-and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair,which in turn promotes the sensitivity of poly(ADP-ribose)polymerase inhibitor(PARPi)in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.展开更多
As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identi...As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.展开更多
Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most ...Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most convenient multicellular organisms for genetic analysis, and the application of this novel genome editing technique to this organism promises to revolutionize analysis of gene function in the future. CRISPR-Cas9 has been successfully used to generate imprecise insertions and deletions via non-homologous end-joining mechanisms and to create precise mutations by homology-directed repair from donor templates. Key variables are the methods used to deliver the Cas9 endonuclease and the efficiency of the single guide RNAs. CRISPR-Cas9-mediated editing appears to be highly specific in C. elegans, with no reported off-target effects. In this review, 1 briefly summarize recent progress in CRISPR-Cas9-based genome editing in C. elegans, highlighting technical improvements in mutagenesis and mutation detection, and discuss potential future appli- cations of this technique.展开更多
It is unknown whether the ss DNA-binding-protein(SSB) possesses the ability to catalyze DNA recombination.We investigated the recombination function of SSB and the recombination kinetics of Escherichia coli using a ne...It is unknown whether the ss DNA-binding-protein(SSB) possesses the ability to catalyze DNA recombination.We investigated the recombination function of SSB and the recombination kinetics of Escherichia coli using a new transformation method with a modified double-layered plate.We found that SSB catalysed intermolecular recombination in vitro.Its intermolecular recombination rate versus substrate concentration or homologous sequence length fitted the Hill equation,and while the plasmid intramolecular recombination rate versus substrate concentration fitted a positively linear correlation,the dominant intermolecular recombination was a non-homologous recombination in vivo,similar to Rec A.However,ssb-dependent recombination occurred later and at a lower recombination rate than the rec A-dependent,probably because ssb expression was about two-fold lower than rec A during the E.coli earlier growth stage.The affinity to substrate and the recombination efficiency of SSB was lower than Rec A,whereas SSB enhanced the catalytic efficiency of Rec A.Knocking out both rec A and ssb led to loss of recombination.Our results confirmed that as SSB has the recombination function itself as an allosteric enzyme,rec Aindependent recombination in E.coli should be ssb-dependent.ssb-dependent recombination may be the third DNA double-strand break repair pathway,in addition to rec Adependent recombination and non-homologous end joining.展开更多
More than half of cancer patients are treated with radiotherapy,which kills tumor cells by directly and indirectly inducing DNA damage,including cytotoxic DNA double-strand breaks(DSBs).Tumor cells respond to these th...More than half of cancer patients are treated with radiotherapy,which kills tumor cells by directly and indirectly inducing DNA damage,including cytotoxic DNA double-strand breaks(DSBs).Tumor cells respond to these threats by activating a complex signaling network termed the DNA damage response(DDR).The DDR arrests the cell cycle,upregulates DNA repair,and triggers apoptosis when damage is excessive.The DDR signaling and DNA repair pathways are fertile terrain for therapeutic intervention.This review highlights strategies to improve therapeutic gain by targeting DDR and DNA repair pathways to radiosensitize tumor cells,overcome intrinsic and acquired tumor radioresistance,and protect normal tissue.Many biological and environmental factors determine tumor and normal cell responses to ionizing radiation and genotoxic chemotherapeutics.These include cell type and cell cycle phase distribution;tissue/tumor microenvironment and oxygen levels;DNA damage load and quality;DNA repair capacity;and susceptibility to apoptosis or other active or passive cell death pathways.We provide an overview of radiobiological parameters associated with X-ray,proton,and carbon ion radiotherapy;DNA repair and DNA damage signaling pathways;and other factors that regulate tumor and normal cell responses to radiation.We then focus on recent studies exploiting DSB repair pathways to enhance radiotherapy therapeutic gain.展开更多
基金supported by Major Program of the National Natural Science Foundation of China(21621004)the Natural Science Foundation of Tianjin City(19JCQNJC09200)the Young Elite Scientists Sponsorship Program by Tianjin(TJSQNTJ-2018-16).
文摘Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.
基金supported by the grants from the Natural Science Foundation of China (No.81201181 to F.G.81473295 and 81670882 to Z.M.S and 81700885 to X.L.G.)+5 种基金Zhejiang Provincial & Ministry of Health research fund for medical sciences (WKJ2013-2-023 to F.G.WKJ-ZJ-1828 to J.Z.Z.and 2016KYA145 to X.L.G.)Science Technology Project of Zhejiang Province (2014C33260 to Z.M.S.and 2017C37176 to F.G.)Eye Hospital at Wenzhou Medical University (YNZD201602 to F.G.)Wenzhou City (Y20160008 to J.Z.Z.)Research Fund for Lin He's Academician Workstation of New Medicine and Clinical Translation (17331209 to C.B.L.)
文摘CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells(Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014).The most widely used customized CRISPR/Cas9(Sp Cas9)is derived from Streptococcus pyogenes(Cong et al.,2013).
基金This work was supported by grants from the National Natural Science Foundation of China[31730003,31670077]Natural Science Foundation of Shandong Province[ZR2017ZB0210].
文摘DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.
基金supported by the National Science and Technology Major Project for Breeding of New Transgenic Organisms, China (2016ZX08006002)the Guangdong Province "Flying Sail Program" Postdoctoral Foundation, China (2016)
文摘Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.
基金Supported by The Grants from the Ministry of EducationCulture+7 种基金SportsScience and Technology of Japanthe Ministry of HealthLabour and Welfare of Japanthe National Institute of Biomedical Innovationthe Asahi Glass Foundationthe Ichiro Kanehara Foundationthe Program for Cultivating Global Leaders in Heavy Ion Therapeutics and Engineering
文摘Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.
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.
文摘The Szent-Gyorgyi Prize for Progress in Cancer Research is a prestigious scientific award established by the National Foundation for Cancer Research(NFCR)—a leading cancer research charitable organization in the United States that is committed to supporting scientific research and public education relating to the prevention,early diagnosis,better treatments,and ultimately,a cure for cancer.Each year,the Szent-Gyorgyi Prize honors an outstanding researcher,nominated by colleagues or peers,who has contributed outstanding,significant research to the fight against cancer,and whose accomplishments have helped improve treatment options for cancer patients.The Prize also promotes public awareness of the importance of basic cancer research and encourages the sustained investment needed to accelerate the translation of these research discoveries into new cancer treatments.This report highlights the pioneering work led by the 2015 Prize winner,Dr.Frederick Alt.Dr.Alt's work in the area of cancer genetics over four decades has helped to shape the very roots of modern cancer research.His work continues to profoundly impact the approaches that doctors around the globe use to diagnose and treat cancer.In particular,his seminal discoveries of gene amplification and his pioneering work on molecular mechanisms of DNA damage repair have helped to usher in the era of genetically targeted therapy and personalized medicine.
基金Supported by The DOD Innovator and Scholar Concept Award,No.W81XWH-12-1-0372
文摘Over the past few decades, major strides have advanced the techniques for early detection and treatment of cancer. However, metastatic tumor growth still accounts for the majority of cancer-related deaths worldwide. In fact, breast cancers are notorious for relapsing years or decades after the initial clinical treatment, and this relapse can vary according to the type of breast cancer. In estrogen receptor-positive breast cancers, late tumor relapses frequently occur whereas relapses in estrogen receptor-negative cancers or triple negative tumors arise early resulting in a higher mortality risk. One of the main causes of metastasis is tumor dormancy in which cancer cells remain concealed, asymptomatic, and untraceable over a prolonged period of time. Under certain conditions, dormant cells can re-enter into the cell cycle and resume proliferation leading to recurrence. However, the molecular and cellular regulators underlying this transition remain poorly understood. To date, three mechanisms have been identified to trigger tumor dormancy including cellular, angiogenic, and immunologic dormancies. In addition, recent studies have suggested that DNA repair mechanisms may contribute to the survival of dormant cancer cells. In this article, we summarize the recent experimental and clinical evidence governing cancer dormancy. In addition, we will discuss the role of DNA repair mechanisms in promoting the survival of dormant cells. This information provides mechanistic insight to explain why recurrence occurs, and strategies that may enhance therapeutic approaches to prevent disease recurrence.
文摘It is proposed that mismatch repair (MMR) mediates the cytotoxic effects of DNA damaging agents by exerting a futile repair pathway which leads to double strand breaks (DSBs). Previous reports indicate that the sensitivity of cells defective in homologous recombination (HR) to DNA alkylation is reduced by defects in MMR genes. We have assessed the contribution of different MMR genes to the processing of alkylation damage in vivo. We have directly visualized recombination complexes formed upon DNA damage using fluorescent protein (FP) fusions. We find that msh6 mutants are more resistant than wild type cells to MNNG, and that an msh6 mutation rescues the sensitivity of rad52 strains more efficiently than an msh3 mutation. Analysis of RAD52-GFP tagged strains indicate that MNNG increases repair foci formation, and that the inactivation of the MHS2 and MSH6 genes but not the MSH3 gene result in a reduction of the number of foci formed. In addition, in the absence of HR, NHEJ could process the MNNG-induced DSBs as indicated by the formation of NHEJ-GFP tagged foci. These data suggest that processing of the alkylation damage by MMR, mainly by MSH2-MSH6, is required for recruitment of recombination proteins to the damage site for repair.
基金supported by grants from the National Basic Research Program of China (973 program, 2012CB113900)the National Natural Science Foundation of China (31071802)the Chongqing Natural Science Foundation (2011BA1002)
文摘Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.
基金supported by the National Natural Science Foundation of China (Nos. 91749115 and 81872298)the Natural Science Foundation of Jiangxi Province (No. 20181BAB205044), China。
文摘DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks(DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination(HR) and non-homologous end joining(NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair.Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes(DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.
基金supported by the National Key Research and Development Program of China(No.2018YFC2000100)the National Natural Science Foundation of China(Nos.31730021,31971220,and 31961160725)+1 种基金the Fok Ying Tung Education Foundationthe China’s Fundamental Research Funds for the Central Universities。
文摘Genome stability can be threatened by both endogenous and exogenous agents.Organisms have evolved numerous mechanisms to repair DNA damage,including homologous recombination(HR)and non-homologous end joining(NHEJ).Among the factors associated with DNA repair,the MRE11-RAD50-NBS1(MRN)complex(MRE11-RAD50-XRS2 in Saccharomyces cerevisiae)plays important roles not only in DNA damage recognition and signaling but also in subsequent HR or NHEJ repair.Upon detecting DNA damage,the MRN complex activates signaling molecules,such as the protein kinase ataxia-telangiectasia mutated(ATM),to trigger a broad DNA damage response,including cell cycle arrest.The nuclease activity of the MRN complex is responsible for DNA end resection,which guides DNA repair to HR in the presence of sister chromatids.The MRN complex is also involved in NHEJ,and has a species-specific role in hairpin repair.This review focuses on the structure of the MRN complex and its function in DNA damage repair.
文摘Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break(DSB)signaling.P53-binding protein 1(53BP1)plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining(NHEJ)-mediated DSB repair pathway that rejoins DSB ends.New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination(HR)signaling.This review focuses on the up-and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair,which in turn promotes the sensitivity of poly(ADP-ribose)polymerase inhibitor(PARPi)in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.
文摘As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.
基金supported by National Institutes of Health (NIH grant R01 GM054657) to A.D.C
文摘Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most convenient multicellular organisms for genetic analysis, and the application of this novel genome editing technique to this organism promises to revolutionize analysis of gene function in the future. CRISPR-Cas9 has been successfully used to generate imprecise insertions and deletions via non-homologous end-joining mechanisms and to create precise mutations by homology-directed repair from donor templates. Key variables are the methods used to deliver the Cas9 endonuclease and the efficiency of the single guide RNAs. CRISPR-Cas9-mediated editing appears to be highly specific in C. elegans, with no reported off-target effects. In this review, 1 briefly summarize recent progress in CRISPR-Cas9-based genome editing in C. elegans, highlighting technical improvements in mutagenesis and mutation detection, and discuss potential future appli- cations of this technique.
基金supported by the Natural Science Foundation of Henan Province (112300410115)the Natural Science Key Foundation of Department of Education (Henan Province)[13A180483]the Program for Innovative Research Team from Henan Province,China (15IRTSTHN014)
文摘It is unknown whether the ss DNA-binding-protein(SSB) possesses the ability to catalyze DNA recombination.We investigated the recombination function of SSB and the recombination kinetics of Escherichia coli using a new transformation method with a modified double-layered plate.We found that SSB catalysed intermolecular recombination in vitro.Its intermolecular recombination rate versus substrate concentration or homologous sequence length fitted the Hill equation,and while the plasmid intramolecular recombination rate versus substrate concentration fitted a positively linear correlation,the dominant intermolecular recombination was a non-homologous recombination in vivo,similar to Rec A.However,ssb-dependent recombination occurred later and at a lower recombination rate than the rec A-dependent,probably because ssb expression was about two-fold lower than rec A during the E.coli earlier growth stage.The affinity to substrate and the recombination efficiency of SSB was lower than Rec A,whereas SSB enhanced the catalytic efficiency of Rec A.Knocking out both rec A and ssb led to loss of recombination.Our results confirmed that as SSB has the recombination function itself as an allosteric enzyme,rec Aindependent recombination in E.coli should be ssb-dependent.ssb-dependent recombination may be the third DNA double-strand break repair pathway,in addition to rec Adependent recombination and non-homologous end joining.
文摘More than half of cancer patients are treated with radiotherapy,which kills tumor cells by directly and indirectly inducing DNA damage,including cytotoxic DNA double-strand breaks(DSBs).Tumor cells respond to these threats by activating a complex signaling network termed the DNA damage response(DDR).The DDR arrests the cell cycle,upregulates DNA repair,and triggers apoptosis when damage is excessive.The DDR signaling and DNA repair pathways are fertile terrain for therapeutic intervention.This review highlights strategies to improve therapeutic gain by targeting DDR and DNA repair pathways to radiosensitize tumor cells,overcome intrinsic and acquired tumor radioresistance,and protect normal tissue.Many biological and environmental factors determine tumor and normal cell responses to ionizing radiation and genotoxic chemotherapeutics.These include cell type and cell cycle phase distribution;tissue/tumor microenvironment and oxygen levels;DNA damage load and quality;DNA repair capacity;and susceptibility to apoptosis or other active or passive cell death pathways.We provide an overview of radiobiological parameters associated with X-ray,proton,and carbon ion radiotherapy;DNA repair and DNA damage signaling pathways;and other factors that regulate tumor and normal cell responses to radiation.We then focus on recent studies exploiting DSB repair pathways to enhance radiotherapy therapeutic gain.