Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica

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.

Functional non-homologous end joining patterns triggered by CRISPR/Cas9 in human cells

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）.

The phage T4 DNA ligase mediates bacterial chromosome DSBs repair as single component non-homologous end joining

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.

DNA end binding activity and Ku70/80 heterodimer expression in human colorectal tumor

AIM： TO determine the DNA binding activity and protein levels of the Ku70/80 heterodimer, the functional mediator of the NHEJ activity, in human colorectal carcinogenesis. METHODS： The Ku70/80 DNA-binding activity was determined by electrophoretic mobility shift assays in 20 colon adenoma and 15 colorectal cancer samples as well as matched normal colonic tissues. Nuclear and cytoplasmic protein expression was determined by immunohistochemistry and Western blot analysis. RESULTS： A statistical found in both adenomas y significant difference was and carcinomas as compared to matched normal colonic mucosa （P〈0.00）. However, changes in binding activity were not homogenous with approximately 50% of the tumors showing a clear increase in the binding activity, 30% displaying a modest increase and 15% showing a decrease of the activity.Tumors, with increased DNA-binding activity, also showed a statistically significant increase in Ku70 and Ku86 nuclear expression, as determined by Western blot and immunohistochemical analyses （P〈0.001）. Cytoplasmic protein expression was found in pathological samples, but not in normal tissues either from tumor patients or from healthy subjects. CONCLUSION： Overall, our DNA-binding activity and protein level are consistent with a substantial activation of the NHEJ pathway in colorectal tumors. Since the NHEJ is an error prone mechanism, its abnormal activation can result in chromosomal instability and ultimately lead to tumorigenesis.

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.

Genome engineering using the CRISPR/Cas system

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.

Frederick W.Alt received the 2015 Szent-Gyrgi Prize for Progress in Cancer Research

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.

New insights into tumor dormancy:Targeting DNA repair pathways

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.

The Role of DNA Mismatch Repair and Recombination in the Processing of DNA Alkylating Damage in Living Yeast Cells

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.

Analysis of Up-Regulation of DNA-PKcs and Its Mechanism in Human Gliomas

OBJECTIVE To detect the differences in gene expression of nonhomologous end-joining pathway including Ku70, Ku80, ERCC4, lig4 and DNA-PKcs between human primary gliomas and normal brain tissues, and furthermore, to explore the underlying mechanism for the expression alteration.METHODS The expression levels of Ku70, Ku80, ERCC4, lig4 and DNA-PKcs in 36 specimens of glioma and 12 specimens of normal brain tissue were measured using SYBR green-based real- time quantitative PCR. Methylation of DNA-PKcs was detected through methylation-specific PCR （MSP）.RESULTS There was no significant difference in expression of Ku70, Ku80, ERCC4 and lig4 between human primary gliomas and normal brain tissues （P 〈 0.05）, while DNA-PKcs were significantly up-regulated （P = 0.002）. The expression of DNA- PKcs was significantly higher in patients with grade III and IV diseases compared to patients with grade II disease or in normal brain tissues （P 〈 0.05）. Moreover, glioma tissue showed weaker methvlation than normal brain tissue.CONCLUSION The up-regulation of the DNA-PKcs may be associated with pathogenesis of glioma. Demethylation of DNA- PKcs promoter is an important reason for its up-regulation.

Di-and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair

Histone H3 Lys36(H3K36)methylation and its associated modifiers are crucial for DNA double-strand break(DSB)repair,but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear.Here,we unveil the distinct roles of H3K36 dimethylation(H3K36me2)and H3K36 trimethylation(H3K36me3)in DSB repair via non-homologous end joining(NHEJ)or homologous recombination(HR).Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency.y Ku70 and Rfa1 bind H3K36me2-or H3K36me3-modified peptides and chromatin,respectively.Disrupting these interactions impairs y Ku70 and Rfa1 recruitment to damaged H3K36me2-or H3K36me3-rich loci,increasing DNA damage sensitivity and decreasing repair efficiency.Conversely,H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit y Ku70 or Rfa1 under DSB stress.Importantly,human KU70 and RPA1,the homologs of y Ku70 and Rfa1,exclusively associate with H3K36me2 and H3K36me3 in a conserved manner.These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways,highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

cGAS guards against chromosome endto-end fusions during mitosis and facilitates replicative senescence

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.

Role of deubiquitinating enzymes in DNA double-strand break repair

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.

MRN complex is an essential effector of DNA damage repair

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.

CDYL1 fosters double-strand break-induced transcription silencing and promotes homology-directed repair

Cells have evolved DNA damage response （DDR） to repair DNA lesions and thus preserving genomic stability and impeding carcinogenesis. DNA damage induction is accompanied by transient transcription repression. Here, we describe a previously unrecognized role of chromodomain Y-like （CDYL1） protein in fortifying double-strand break （DSB）-induced transcription repression and repair. We showed that CDYL1 is rapidly recruited to damaged euchromatic regions in a poly （ADP-ribose） polymerase 1 （PARP1）-dependent, but ataxia telangiectasia mutated （ATM）-independent, manner. While the C-terminal region, containing the enoyl-CoA hydratase like （ECH） domain, of CDYL1 binds to poly （ADP-ribose） （PAR） moieties and mediates CDYL1 accumulation at DNA damage sites, the chromodomain and histone H3 trimethylated on lysine 9 （H3K9me3） mark are dispensable for its recruitment. Furthermore, CDYL1 promotes the recruitment of enhancer of zeste homolog 2 （EZH2）, stimulates local increase of the repressive methyl mark H3K27me3, and promotes transcription silencing at DSB sites. In addition, following DNA damage induction, CDYL1 depletion causes persistent G2/M arrest and alters H2AX and replication protein A （RPA2） phosphorylation. Remarkably, the ‘traffic-light reporter’ system revealed that CDYL1 mainly promotes homology-directed repair （HDR） of DSBs in vivo. Consequently, CDYL1-knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin. Altogether, our findings identify CDYL1 as a new component of the DDR and suggest that the HDR-defective ‘BRCAness’ phenotype of CDYL1-deficient cells could be exploited for eradicating cancer cells harboring CDYL1 mutations.

联合抑制聚腺苷酸二磷酸核糖转移酶-1及DNA依赖蛋白激酶催化亚单位增加KG-1α细胞对依托泊苷的敏感性

目的观察联合抑制聚腺苷酸二磷酸核糖转移酶-1[poly(ADP-ribose)polymerase-1,PARP-1]及DNA依赖蛋白激酶催化亚单位(catalytic sunbunit of the DNA-dependent protein kinase,DNA-PKcs)对依托泊苷(etoposide,VP-16)作用于急性髓系白血病KG-1α细胞的影响。方法分别用特异性抑制剂奥拉帕尼(Olaparib,OLA)和Nu7441(NU)抑制PARP-1、DNA-PKcs。将KG-1α细胞设置成对照组、模型组、PARP-1抑制组(PARP-1 inhibition,VP-16+OLA)、DNA-PKcs抑制组(DNA-PKcs inhibition,VP-16+NU)、联合抑制组(combined inhibition,VP-16+OLA+NU)。用四甲基偶氮唑蓝(MTT)法体外检测联合用药对KG-1α细胞增殖抑制的影响;用高内涵荧光成像检测分析不同处理组的γ-H2AX在DNA双链断裂断裂位点的募集情况;用蛋白质印迹法检测细胞凋亡相关蛋白Cleaved caspase-3和Cleaved PARP的表达情况。结果对照组、模型组、PARP-1抑制组、DNA-PKcs抑制组和联合抑制组的增殖抑制率分别为(1.16±1.45)%,(23.88±3.15)%,(28.12±2.28)%,(17.21±0.89)%和(60.72±4.38)%;γ-H2AX阳性细胞百分比分别为γ-H2 AX阳性细胞百分比分别为(1.24±0.07)%,(22.51±2.24)%,(24.55±3.29)%,(23.28±2.48)%和(40.55±1.61)%;Cleaved Caspase-3蛋白相对表达量分别为0.16±0.10,0.61±0.30,1.15±0.64,1.11±0.31和1.72±1.03;Cleaved PARP蛋白相对表达量分别为1.23±0.09,1.45±0.55,2.03±0.84,2.08±0.41和2.66±0.95。模型组分别与对照组、联合抑制组比较,差异均有统计学意义(均P<0.05)。结论联合抑制PARP-1及DNA-PKcs能够在体外增加KG-1α细胞对依托泊苷的敏感性。