Objective:The choice of chemotherapeutic regimen for triple-negative breast cancer(TNBC)remains controversial.Homologous recombination deficiency(HRD)has attracted increasing attention in informing chemotherapy treatm...Objective:The choice of chemotherapeutic regimen for triple-negative breast cancer(TNBC)remains controversial.Homologous recombination deficiency(HRD)has attracted increasing attention in informing chemotherapy treatment.This study was aimed at investigating the feasibility of HRD as a clinically actionable biomarker for platinum-containing and platinum-free therapy.Methods:Chinese patients with TNBC who received chemotherapy between May 1,2008 and March 31,2020 were retrospectively analyzed with a customized 3D-HRD panel.HRD positivity was defined by an HRD score≥30 or deleterious BRCA1/2 mutation.A total of 386 chemotherapy-treated patients with TNBC were screened from a surgical cohort(NCT01150513)and a metastatic cohort,and 189 patients with available clinical and tumor sequencing data were included.Results:In the entire cohort,49.2%(93/189)of patients were identified as HRD positive(40 with deleterious BRCA1/2 mutations and 53 with BRCA1/2 intact with an HRD score of≥30).In the first-line metastatic setting,platinum therapy was associated with longer median progression-free survival(mPFS)than platinum-free therapy[9.1 vs.3.0 months;hazard ratio(HR),0.43;95%confidence interval 0.22–0.84;P=0.01].Among HRD-positive patients,the mPFS was significantly longer in those treated with platinum rather than platinum-free therapy(13.6 vs.2.0 months;HR,0.11;P=0.001).Among patients administered a platinum-free regimen,HRD-negative patients showed a PFS significantly superior to that of HRD-positive patients(P=0.02;treatment-biomarker P-interaction=0.001).Similar results were observed in the BRCA1/2-intact subset.In the adjuvant setting,HRD-positive patients tended to benefit more from platinum chemotherapy than from platinum-free chemotherapy(P=0.05,P-interaction=0.02).Conclusions:HRD characterization may guide decision-making regarding the use of platinum treatment in patients with TNBC in both adjuvant and metastatic settings.展开更多
Objective:Currently,there is an urgent need to identify immunotherapeutic biomarkers to increase the benefit of immune checkpoint inhibitors(ICIs)for patients with gastric cancer(GC).Homologous recombination deficienc...Objective:Currently,there is an urgent need to identify immunotherapeutic biomarkers to increase the benefit of immune checkpoint inhibitors(ICIs)for patients with gastric cancer(GC).Homologous recombination deficiency(HRD)can modify the tumor immune microenvironment by increasing the presence of tumor-infiltrating lymphocytes and therefore might serve as a biomarker of immunotherapeutic response.We aimed to analyze the mutational pattern of HR-associated genes in Chinese patients with GC and its relevance to the tumor immune profile and clinical immunotherapeutic response.Methods:A panel of 543 cancer-associated genes was used to analyze genomic profiles in a cohort comprising 484 Chinese patients with GC.Correlations between HR gene mutations and tumor immunity or clinical outcomes were identified via bioinformatic analysis using 2 GC genomic datasets(TCGA and MSK-IMPACT).Results:Fifty-one of the 484(10.54%)patients carried at least one somatic mutation in an HR gene;ATM(16/484,3.31%)was among the most frequently mutated HR genes in the Chinese cohort.Mutations in HR genes were associated with elevated tumor mutational burden,enhanced immune activity,and microsatellite instability status.In the MSK-IMPACT cohort comprising 49 patients with stomach adenocarcinoma or gastroesophageal junction adenocarcinoma treated with ICIs,patients with HR-mut GC(n=12)had significantly better overall survival than those with HR-wt GC(n=37)(log-rank test,P<0.05).Conclusions:Our data suggest that detection of somatic mutations in HR genes might aid in identifying patients who might benefit from immune checkpoint blockade therapy.展开更多
Objective:We aimed to investigate the radiosensitizing efficacy of the poly-ADP-ribose polymerase(PARP)inhibitor,olaparib,and the Bloom syndrome protein(BLM)helicase inhibitor,ML216,in non-small cell lung cancer(NSCLC...Objective:We aimed to investigate the radiosensitizing efficacy of the poly-ADP-ribose polymerase(PARP)inhibitor,olaparib,and the Bloom syndrome protein(BLM)helicase inhibitor,ML216,in non-small cell lung cancer(NSCLC)cells.Methods:Radiosensitization of NSCLC cells was assessed by colony formation and tumor growth assays.Mechanistically,the effects of ML216,olaparib,and radiation on cell and tumor proliferation,DNA damage,cell cycle,apoptosis,homologous recombination(HR)repair,and non-homologous end joining(NHEJ)repair activity were determined.Results:Both olaparib and ML216 enhanced the radiosensitivities of olaparib-sensitive H460 and H1299 cells,which was seen as decreased surviving fractions and Rad51 foci,increased total DNA damage,andγH2AX and 53BP1 foci(P<0.05).The expressions of HR repair proteins were remarkably decreased in olaparib-treated H460 and H1299 cells after irradiation(P<0.05),while olaparib combined with ML216 exerted a synergistic radiosensitization effect on olaparib-resistant A549 cells.In addition to increases of double strand break(DSB)damage and decreases of Rad51 foci,olaparib combined with ML216 also increased pDNA-PKcs(S2056)foci,abrogated G2 cell cycle arrest,and induced apoptosis in A549 lung cancer after irradiation in vitro and in vivo(P<0.05).Moreover,Western blot showed that olaparib combined with ML216 and irradiation inhibited HR repair,promoted NHEJ repair,and inactivated cell cycle checkpoint signals both in vitro and in vivo(P<0.05).Conclusions:Taken together,these results showed the efficacy of PARP and BLM helicase inhibitors for radiosensitizing NSCLC cells,and supported the model that BLM inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization,as well as providing the basis for the potential clinical development of this combination for tumors intrinsically resistant to PARP inhibitors and radiotherapy.展开更多
Japanese encephalitis virus(JEV) is a significant causative agent of arthropod-borne encephalitis and what is less clear that the factors cause the virus wide spread. The objective was to confirm whether the homologou...Japanese encephalitis virus(JEV) is a significant causative agent of arthropod-borne encephalitis and what is less clear that the factors cause the virus wide spread. The objective was to confirm whether the homologous recombination imposed on JEV. The phylogenetic and homologous recombination analyses were performed based on 163 complete JEV genomes which were recently isolated. They were still separated into five genotypes(GI-GV) and the most of recently isolated JEVs were GI rather than GIII in Asian areas including China's Mainland. Two recombinant events were identified in JEV and the evidence of the recombination was observed between China and Japan isolates that partitioned into two distinct subclades, but still the same genotype(GIII). Our data further suggested that most of the nucleotides in JEV genome were under negative selection; however, changes within codon 2 316(amino acid NS4b-44) showed an evidence of the positive selection.展开更多
Latency is the pivotal factor that governs the long-term pathogenecity and persistence of HIV-1 infection.It is also the primary impediment to cure and successful treatment,resulting in patient death.Latency of HIV-1 ...Latency is the pivotal factor that governs the long-term pathogenecity and persistence of HIV-1 infection.It is also the primary impediment to cure and successful treatment,resulting in patient death.Latency of HIV-1 infection promotes failure of the conventional antiretroviral therapy(ART).Cessation of ART immediately leads to viral reactivation and attainment of viral load in peripheral circulation.ART comes with severe side effects and is ineffective at treating latent infection.To eliminate latent infection,alternate therapeutic strategies such as Shock and Kill,Block and Lock,gene editing and vaccination have been proposed.Although these strategies have experimentally been proven successful,they possess major limitations.Hence,in this hypothesis,an alternate therapeutic strategy has been proposed to solve the threat of HIV-latency.The proposed model encompasses the generation and administration of recombinant HIV-1 particles whose genomes having pro-apoptotic gene,tBid,will activate apoptotic cell death pathways after infecting only the latent cells,thereby removing latent HIV host cells from patients’bodies.展开更多
Defects in genes involved in the DNA damage response cause homologous recombination repair deficiency(HRD).HRD is found in a subgroup of cancer patients for several tumor types,and it has a clinical relevance to cance...Defects in genes involved in the DNA damage response cause homologous recombination repair deficiency(HRD).HRD is found in a subgroup of cancer patients for several tumor types,and it has a clinical relevance to cancer prevention and therapies.Accumulating evidence has identified HRD as a biomarker for assessing the therapeutic response of tumor cells to poly(ADP-ribose)polymerase inhibitors and platinum-based chemotherapies.Nevertheless,the biology of HRD is complex,and its applications and the benefits of different HRD biomarker assays are controversial.This is primarily due to inconsistencies in HRD assessments and definitions(gene-level tests,genomic scars,mutational signatures,or a combination of these methods)and difficulties in assessing the contribution of each genomic event.Therefore,we aim to review the biological rationale and clinical evidence of HRD as a biomarker.This review provides a blueprint for the standardization and harmonization of HRD assessments.展开更多
Background:It is of great clinical significance to further explore new strategies and potential combined therapeutic targets for gastric cancer.This study aimed to investigate the synthetic lethal effect of RBBP8 mole...Background:It is of great clinical significance to further explore new strategies and potential combined therapeutic targets for gastric cancer.This study aimed to investigate the synthetic lethal effect of RBBP8 molecular intervention combined with a poly ADP ribose polymerase(PARP)inhibitor in non-BRCA mutant gastric cancer and clarify the mechanism by which RBBP8 regulates homologous recombination repair.Methods:The role of RBBP8 in DNA damage repair was observed using bioinformatic analysis,western blot analysis,and immunofluorescence.The synthetic lethal effect was verified using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt(MTS)and flow cytometry apoptosis experiments.Results:Among the patients with gastric cancer treated with chemotherapy,the prognosis of patients with high RBBP8 expression levels was worse(homologous recombination[HR]=1.54,p=0.028).RBBP8 knockdown induced DNA damage and had a synergistic effect with PARP inhibitor treatment on cell viability inhibition and cell apoptosis in AGS(generic code for human gastric adenocarcinoma cells)(t=11.154,p<0.001)and N87(t=6.362,p<0.001)cells.RBBP8 knockdown inhibited RAD51 activation and DNA terminal excision in homologous recombination repair.Conclusion:RBBP8 is involved in homologous recombination repair,and molecular intervention into RBBP8 could achieve a synthetic lethal effect with PARP inhibitor treatment in gastric cancer cells.展开更多
Ovarian cancer is the most lethal gynecologic cancer. Optimal cytoreductive surgery followed by platinum-based chemotherapy with or without bevacizumab is the conventional therapeutic strategy. Since 2016, the pharmac...Ovarian cancer is the most lethal gynecologic cancer. Optimal cytoreductive surgery followed by platinum-based chemotherapy with or without bevacizumab is the conventional therapeutic strategy. Since 2016, the pharmacological treatment of epithelial ovarian cancer has significantly changed following the introduction of the poly (ADP-ribose) polymerase inhibitors (PARPi). BRCA1/2 mutations and homologous recombination deficiency (HRD) have been established as predictive biomarkers of the benefit from platinum-based chemotherapy and PARPi. While in the absence of HRD (the so-called homologous recombination proficiency, HRp), patients derive minimal benefit from PARPi, the use of the antiangiogenic agent bevacizumab in first line did not result in different efficacy according to the presence of homologous recombination repair (HRR) genes mutations. No clinical trials have currently compared PARPi and bevacizumab as maintenance therapy in the HRp population. Different strategies are under investigation to overcome primary and acquired resistance to PARPi and to increase the sensitivity of HRp tumors to these agents. These tumors are characterized by frequent amplifications of Cyclin E and MYC, resulting in high replication stress. Different agents targeting DNA replication stress, such as ATR, WEE1 and CHK1 inhibitors, are currently being explored in preclinical models and clinical trials and have shown promising preliminary signs of activity. In this review, we will summarize the available evidence on the activity of PARPi in HRp tumors and the ongoing research to develop new treatment options in this hard-to-treat population.展开更多
Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hF...Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.展开更多
Divalent metal transporter 1(DMT1)is a ferrous iron import protein.The improper expression of DMT1 is involved in neurodegenerative diseases.In the present study,we constructed a recombinant adenovirus containing the ...Divalent metal transporter 1(DMT1)is a ferrous iron import protein.The improper expression of DMT1 is involved in neurodegenerative diseases.In the present study,we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element(DMT1-IRE)and investigated its expression and function in the C6 glioma cell line.The DMT1-IRE gene,obtained by RT-PCR,was cloned into the shuttle plasmid-ing pAdTrack-CMV containing greenfluorescent protein(GFP)reporter gene.Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escher-ichia coli(E.coli)BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I.Pac I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells(HEK293 cells),in which recombinant adenoviruses were generated within 7 to 10 days.The results demon-strated that we obtained the DMT1-IRE gene.pAdEasy1-DMT1-IRE yielded a large fragment,plus a smaller fragment of 4.5 kb after digestion with Pac I.PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE,indicating the successful construction of recombi-nant adenovirus plasmid containing DMT1-IRE.GFPfluorescence further confirmed the generation of recombi-nant AdDMT1-IRE adenovirus.AdDMT1-IRE could efficiently infect C6 glioma cells.And cell viability decreased in AdDMT1-IRE infected cells after iron overload compared to the control.These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.展开更多
Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplifi...Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.展开更多
Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further inves...Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further investigated the potential association between DNAH2 and the homologous recombination repair pathway of FA.The assays of homologous recombination repair,mitomycin C(MMC)sensitivity,immunofluorescence,and ubiquitination modification were performed in U2OS and DR-U2OS cell lines.In MMC-treated U2OS cells,the downregulation of the DNAH2 gene increased the sensitivity of cells to DNA inter-strand crosslinks.We also observed the reduced enrichment of FANCD2 protein to DNA damage sites.Furthermore,the ubiquitination modification level of FANCD2 was influenced by the deficiency of DNAH2.Thus,our results suggest that DNAH2 may modulate the cell homologous recombination repair partially by increasing the ubiquitination and the enrichment to DNA damage sites of FANCD2.DNAH2 may act as a novel co-pathogenic gene of FA patients.展开更多
Background:Few studies have investigated the characteristics of non‐BRCA homologous recombination repair(HRR)pathway somatic mutations,and the impact of these mutations on efficacy of treatment in ovarian cancer pati...Background:Few studies have investigated the characteristics of non‐BRCA homologous recombination repair(HRR)pathway somatic mutations,and the impact of these mutations on efficacy of treatment in ovarian cancer patients is not clear.Therefore,we conducted this study to analyze the frequency and spectrum of somatic mutations in HRR pathway genes in patients with ovarian cancer and to examine the relationships between somatic mutations in HRR pathway genes and their effects on the efficacy of platinum‐based chemotherapy.Methods:We performed targeted sequencing of 688 genes related to the occurrence,development,treatment,and prognosis of solid tumors.Somatic mutations were identified by paired analysis of tumor tissue and germline DNA in blood cells.Results:A total of 38 patients with ovarian cancer were included in the study,and 35(92.1%)patients were diagnosed with high‐grade serous carcinoma.All patients exhibited somatic mutations in the tumor tissue samples.The commonly mutated genes were TP53(73.7%),BRCA2(55.3%),NF1(52.6%),BRCA1(47.4%),and CDH1(47.4%).Overall,71.1%of the patients exhibited mutation in at least one HRR pathway gene.The most frequently altered HRR genes were BRCA2(55.3%),followed by BRCA1(47.4%),ATM(44.7%),BARD1(42.1%),and CHEK1(36.8%).The median progression‐free survival(PFS)in patients with HRR pathway mutation was 36.0 months compared with 13.6 months in patients with no HRR pathway mutation(hazard ratio[HR],0.25;95%confidence interval[CI],0.08–0.77;p=0.016).Patients harboring BRCA1/2 and/or CDK12 mutations displayed a longer PFS(median,36.0 months)compared with patients with no BRCA1/2 or CDK12 mutation(median,13.6 months;HR,0.21;95%CI,0.07–0.61;p=0.004).In multivariate analysis Cox proportional hazards models,after adjustment for tumor stage at diagnosis and histology of initial diagnosis,patients with HRR pathway mutation had a longer PFS than patients with HRR wild‐type genes(p=0.006).Conclusions:HRR pathway somatic mutations are common in Chinese patients with ovarian cancer.HRR pathway somatic mutations were associated with improved sensitivity to platinum-based chemotherapy.Large-scale prospective studies are needed to verify our findings.展开更多
Phage-encoded homologous recombination(PEHR)is an efficient tool for bacterial genome editing.We previously developed and utilized a Pseudomonas-specific PEHR system.However,when using the PEHR system for Pseu-domonas...Phage-encoded homologous recombination(PEHR)is an efficient tool for bacterial genome editing.We previously developed and utilized a Pseudomonas-specific PEHR system.However,when using the PEHR system for Pseu-domonas genome editing,false positives can be a problem.In this study,we combined a compact Cascade-Cas3 system from P.aeruginosa(PaeCas3c)with a Pseudomonas-specific PEHR system,and the results of our recom-bineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy.展开更多
Whether prokaryotes,and other microorganisms,form distinct clusters that can be recognized as species remains an issue of paramount theoretical as well as practical consequence in identifying,regulating,and communicat...Whether prokaryotes,and other microorganisms,form distinct clusters that can be recognized as species remains an issue of paramount theoretical as well as practical consequence in identifying,regulating,and communicating about these organisms.In the past decade,comparisons of thousands of genomes of isolates and hundreds of metagenomes have shown that prokaryotic diversity may be predominantly organized in such sequence-discrete clusters,albeit organisms of intermediate relatedness between the identified clusters are also frequently found.Accumulating evidence suggests,however,that the latter“intermediate”organisms show enough ecological and/or functional distinctiveness to be considered different species.Notably,the area of discontinuity between clusters often—but not always—appears to be around 85%–95%genome-average nucleotide identity,consistently among different taxa.More recent studies have revealed remarkably similar diversity patterns for viruses and microbial eukaryotes as well.This high consistency across taxa implies a specific mechanistic process that underlies the maintenance of the clusters.The underlying mechanism may be a substantial reduction in the efficiency of homologous recombination,which mediates(successful)horizontal gene transfer,around 95%nucleotide identity.Deviations from the 95%threshold(e.g.,species showing lower intraspecies diversity)may be caused by ecological differentiation that imposes barriers to otherwise frequent gene transfer.While this hypothesis that clusters are driven by ecological differentiation coupled to recombination frequency(i.e.,higher recombination within vs.between groups)is appealing,the supporting evidence remains anecdotal.The data needed to rigorously test the hypothesis toward advancing the species concept are also outlined.展开更多
Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primari...Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primarily involved in N^(6)-threonylcarbamoyl adenosine(t^(6)A)modification of transfer RNAs(tRNAs).Recently,it was reported that KEOPS participates in homologous recombination(HR)repair in yeast.To characterize the KEOPS in archaea(aKEOPS),we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus.We show that aKEOPS also possesses five subunits,Pcc1,Kae1,Bud32,Cgi121,and Pcc1-like(or Gon7-like),just like eukaryotic KEOPS.Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex(Kae1-Pcc1-Pcc1-Kae1),suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit.Strikingly,none of the genes encoding aKEOPS subunits,including Pcc1 and Pcc1-like,can be deleted in the wild type and in a t^(6)A modification complementary strain named TsaKI,implying that the aKEOPS complex is essential for an additional cellular process in this archaeon.Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI.These results suggest that aKEOPS plays an essential role independent of the cellular t^(6)A modification level.In addition,archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹCCA tail binding module.Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7.The study also reveals a possible link between the function in t^(6)A modification and the additional function,presumably HR.展开更多
Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic,cephalosporin.In light of their significant medical value,an efficient gene disruption method is ...Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic,cephalosporin.In light of their significant medical value,an efficient gene disruption method is required for the physiological and biochemical studies on this genus of fungi.However,the number of selection markers that can be used for gene targeting is limited,which constrain the genetic analysis of multiple functional genes.In this study,we established a uridine auxotrophy based marker recycling system which achieves scarless gene deletion,and allows the use of the same selection marker in successive transformations in a deep seaderived fungus Acremonium sp.HDN16-126.We identified one homologue of Acremonium chrysogenum pyrG(also as a homologous gene of the yeast URA3)from HDN16-126,designated as pyrG-A1,which can be used as a selection marker on uridine free medium.We then removed pyrG-A1 from HDN16-126 genome via homologous recombination(HR)on MM medium with 5-fluoroortic acid(5-FOA),a chemical that can be converted into a toxin of 5-flurouracil by pyrG-A1 activity,thus generating the HDN16-126-△pyrG mutant strain which showed auxotrophy for uridine but insensitivity to 5-FOA and enabled the use of exogenous pyrG gene as both positive and negative selection marker to achieve the scarless deletion of target DNA fragments.We further applied this marker recycling system to successfully disrupt two target genes pepL(encodes a putative 2OG-Fe(Ⅱ)dioxygenase)and pepM(encodes a putative aldolase)identified from HDN16-126 genome,which are proposed to be functional genes related to 2-aminoisobutyric acid metabolism in fungi.This work is the first application of uridine auxotrophy based scarless gene deletion method in Acremonium species and shows promising potential in assisting sequential genetic analysis of filamentous fungi.展开更多
RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination.However,the specific pathway(s)in which it is involved and the underlining mechanism(s)remain poorly understood.We took ad...RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination.However,the specific pathway(s)in which it is involved and the underlining mechanism(s)remain poorly understood.We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5(dRecQ5)functions in vivo in homologous recombination-mediated double strand break(DSB)repair.We generated null alleles of dRecQ5 using the targeted recombination technique.The mutant animals are homozygous viable,but with growth retardation during development.The mutants are sensitive to both exogenous DSB-inducing treatment,such as gamma-irradiation,and endogenously induced double strand breaks(DSBs)by I-Sce I endonuclease.In the absence of dRecQ5,single strand annealing(SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion,or non-homologous end joining(NHEJ)when inter-chromosomal homologous sequence is unavailable.Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity(LOH)assays.Together,our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.展开更多
Gastrointestinal mucositis is one of the most debilitating side effects of the chemotherapeutic agent irinotecan(CPT-11). Andrographolide, a natural bicyclic diterpenoid lactone, has been reported to possess anti-coli...Gastrointestinal mucositis is one of the most debilitating side effects of the chemotherapeutic agent irinotecan(CPT-11). Andrographolide, a natural bicyclic diterpenoid lactone, has been reported to possess anti-colitis activity. In this study, andrographolide treatment was found to significantly relieve CPT-11-induced colitis in tumor-bearing mice without decreasing the tumor suppression effect of CPT-11. CPT-11 causes DNA damage and the release of double-stranded DNA(ds DNA) from the intestine, leading to cyclic-GMP-AMP synthase(c GAS)-stimulator of interferon genes(STING)-mediated colitis, which was significantly decreased by andrographolide both in vivo and in vitro. Mechanistic studies revealed that andrographolide could promote homologous recombination(HR) repair and downregulate ds DNA-c GAS-STING signaling and contribute to the improvement of CPT-11-induced gastrointestinal mucositis. These results suggest that andrographolide may be a novel agent to relieve gastrointestinal mucositis caused by CPT-11.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
基金granted by Capital’s Funds for Health Improvement and Research(Grant No.2018-2-4023)the National Natural Science Foundation of China(Grant No.82001559)。
文摘Objective:The choice of chemotherapeutic regimen for triple-negative breast cancer(TNBC)remains controversial.Homologous recombination deficiency(HRD)has attracted increasing attention in informing chemotherapy treatment.This study was aimed at investigating the feasibility of HRD as a clinically actionable biomarker for platinum-containing and platinum-free therapy.Methods:Chinese patients with TNBC who received chemotherapy between May 1,2008 and March 31,2020 were retrospectively analyzed with a customized 3D-HRD panel.HRD positivity was defined by an HRD score≥30 or deleterious BRCA1/2 mutation.A total of 386 chemotherapy-treated patients with TNBC were screened from a surgical cohort(NCT01150513)and a metastatic cohort,and 189 patients with available clinical and tumor sequencing data were included.Results:In the entire cohort,49.2%(93/189)of patients were identified as HRD positive(40 with deleterious BRCA1/2 mutations and 53 with BRCA1/2 intact with an HRD score of≥30).In the first-line metastatic setting,platinum therapy was associated with longer median progression-free survival(mPFS)than platinum-free therapy[9.1 vs.3.0 months;hazard ratio(HR),0.43;95%confidence interval 0.22–0.84;P=0.01].Among HRD-positive patients,the mPFS was significantly longer in those treated with platinum rather than platinum-free therapy(13.6 vs.2.0 months;HR,0.11;P=0.001).Among patients administered a platinum-free regimen,HRD-negative patients showed a PFS significantly superior to that of HRD-positive patients(P=0.02;treatment-biomarker P-interaction=0.001).Similar results were observed in the BRCA1/2-intact subset.In the adjuvant setting,HRD-positive patients tended to benefit more from platinum chemotherapy than from platinum-free chemotherapy(P=0.05,P-interaction=0.02).Conclusions:HRD characterization may guide decision-making regarding the use of platinum treatment in patients with TNBC in both adjuvant and metastatic settings.
基金supported by the Youth Fund Project of NSFC(Grant No.81403242)Development Project of Shanghai Peak Disciplines Integrative Medicine(Grant No.20180101)。
文摘Objective:Currently,there is an urgent need to identify immunotherapeutic biomarkers to increase the benefit of immune checkpoint inhibitors(ICIs)for patients with gastric cancer(GC).Homologous recombination deficiency(HRD)can modify the tumor immune microenvironment by increasing the presence of tumor-infiltrating lymphocytes and therefore might serve as a biomarker of immunotherapeutic response.We aimed to analyze the mutational pattern of HR-associated genes in Chinese patients with GC and its relevance to the tumor immune profile and clinical immunotherapeutic response.Methods:A panel of 543 cancer-associated genes was used to analyze genomic profiles in a cohort comprising 484 Chinese patients with GC.Correlations between HR gene mutations and tumor immunity or clinical outcomes were identified via bioinformatic analysis using 2 GC genomic datasets(TCGA and MSK-IMPACT).Results:Fifty-one of the 484(10.54%)patients carried at least one somatic mutation in an HR gene;ATM(16/484,3.31%)was among the most frequently mutated HR genes in the Chinese cohort.Mutations in HR genes were associated with elevated tumor mutational burden,enhanced immune activity,and microsatellite instability status.In the MSK-IMPACT cohort comprising 49 patients with stomach adenocarcinoma or gastroesophageal junction adenocarcinoma treated with ICIs,patients with HR-mut GC(n=12)had significantly better overall survival than those with HR-wt GC(n=37)(log-rank test,P<0.05).Conclusions:Our data suggest that detection of somatic mutations in HR genes might aid in identifying patients who might benefit from immune checkpoint blockade therapy.
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.31670859,81772243,81803172,81803167,31800703,and 31900889)the CAMS Innovation Fund for Medical Science(Grant No.2017-I2M-1-016)+4 种基金the China Postdoctoral Science Foundation(Grant No.2018M630106)the Natural Science Foundation of Tianjin(Grant Nos.18JCYBJC26800,18JCQNJC12300,and 17JCYBJC42700)the Fundamental Research Funds for the Central Universities(Grant No.10023201601602)the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences(Grant Nos.2017-1001-08 and 2018RC310020)the Key R&D Program of Shandong Province(Grant No.2019GSF107056).
文摘Objective:We aimed to investigate the radiosensitizing efficacy of the poly-ADP-ribose polymerase(PARP)inhibitor,olaparib,and the Bloom syndrome protein(BLM)helicase inhibitor,ML216,in non-small cell lung cancer(NSCLC)cells.Methods:Radiosensitization of NSCLC cells was assessed by colony formation and tumor growth assays.Mechanistically,the effects of ML216,olaparib,and radiation on cell and tumor proliferation,DNA damage,cell cycle,apoptosis,homologous recombination(HR)repair,and non-homologous end joining(NHEJ)repair activity were determined.Results:Both olaparib and ML216 enhanced the radiosensitivities of olaparib-sensitive H460 and H1299 cells,which was seen as decreased surviving fractions and Rad51 foci,increased total DNA damage,andγH2AX and 53BP1 foci(P<0.05).The expressions of HR repair proteins were remarkably decreased in olaparib-treated H460 and H1299 cells after irradiation(P<0.05),while olaparib combined with ML216 exerted a synergistic radiosensitization effect on olaparib-resistant A549 cells.In addition to increases of double strand break(DSB)damage and decreases of Rad51 foci,olaparib combined with ML216 also increased pDNA-PKcs(S2056)foci,abrogated G2 cell cycle arrest,and induced apoptosis in A549 lung cancer after irradiation in vitro and in vivo(P<0.05).Moreover,Western blot showed that olaparib combined with ML216 and irradiation inhibited HR repair,promoted NHEJ repair,and inactivated cell cycle checkpoint signals both in vitro and in vivo(P<0.05).Conclusions:Taken together,these results showed the efficacy of PARP and BLM helicase inhibitors for radiosensitizing NSCLC cells,and supported the model that BLM inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization,as well as providing the basis for the potential clinical development of this combination for tumors intrinsically resistant to PARP inhibitors and radiotherapy.
基金Supported by the National Natural Science Foundation of China(31272569,31270187)Projects in the National Science&Technology Pillar Program during 12th Five-year Plan Period(2013BAD12B04)Harbin Science and Technology Bureau(RC2012XK002003)
文摘Japanese encephalitis virus(JEV) is a significant causative agent of arthropod-borne encephalitis and what is less clear that the factors cause the virus wide spread. The objective was to confirm whether the homologous recombination imposed on JEV. The phylogenetic and homologous recombination analyses were performed based on 163 complete JEV genomes which were recently isolated. They were still separated into five genotypes(GI-GV) and the most of recently isolated JEVs were GI rather than GIII in Asian areas including China's Mainland. Two recombinant events were identified in JEV and the evidence of the recombination was observed between China and Japan isolates that partitioned into two distinct subclades, but still the same genotype(GIII). Our data further suggested that most of the nucleotides in JEV genome were under negative selection; however, changes within codon 2 316(amino acid NS4b-44) showed an evidence of the positive selection.
文摘Latency is the pivotal factor that governs the long-term pathogenecity and persistence of HIV-1 infection.It is also the primary impediment to cure and successful treatment,resulting in patient death.Latency of HIV-1 infection promotes failure of the conventional antiretroviral therapy(ART).Cessation of ART immediately leads to viral reactivation and attainment of viral load in peripheral circulation.ART comes with severe side effects and is ineffective at treating latent infection.To eliminate latent infection,alternate therapeutic strategies such as Shock and Kill,Block and Lock,gene editing and vaccination have been proposed.Although these strategies have experimentally been proven successful,they possess major limitations.Hence,in this hypothesis,an alternate therapeutic strategy has been proposed to solve the threat of HIV-latency.The proposed model encompasses the generation and administration of recombinant HIV-1 particles whose genomes having pro-apoptotic gene,tBid,will activate apoptotic cell death pathways after infecting only the latent cells,thereby removing latent HIV host cells from patients’bodies.
基金supported by the National Key R&D Program of China(Grant No.2022YFC2409902)the National Natural Science Foundation of China(Grant No.82172876)+2 种基金the Beijing Nova Program of Science and Technology(Grant No.Z191100001119095)the Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(Grant No.2021-I2M-1-066)the Beijing Hope Run Special Fund of Cancer Foundation of China(Grant No.LC2019L04).
文摘Defects in genes involved in the DNA damage response cause homologous recombination repair deficiency(HRD).HRD is found in a subgroup of cancer patients for several tumor types,and it has a clinical relevance to cancer prevention and therapies.Accumulating evidence has identified HRD as a biomarker for assessing the therapeutic response of tumor cells to poly(ADP-ribose)polymerase inhibitors and platinum-based chemotherapies.Nevertheless,the biology of HRD is complex,and its applications and the benefits of different HRD biomarker assays are controversial.This is primarily due to inconsistencies in HRD assessments and definitions(gene-level tests,genomic scars,mutational signatures,or a combination of these methods)and difficulties in assessing the contribution of each genomic event.Therefore,we aim to review the biological rationale and clinical evidence of HRD as a biomarker.This review provides a blueprint for the standardization and harmonization of HRD assessments.
文摘Background:It is of great clinical significance to further explore new strategies and potential combined therapeutic targets for gastric cancer.This study aimed to investigate the synthetic lethal effect of RBBP8 molecular intervention combined with a poly ADP ribose polymerase(PARP)inhibitor in non-BRCA mutant gastric cancer and clarify the mechanism by which RBBP8 regulates homologous recombination repair.Methods:The role of RBBP8 in DNA damage repair was observed using bioinformatic analysis,western blot analysis,and immunofluorescence.The synthetic lethal effect was verified using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt(MTS)and flow cytometry apoptosis experiments.Results:Among the patients with gastric cancer treated with chemotherapy,the prognosis of patients with high RBBP8 expression levels was worse(homologous recombination[HR]=1.54,p=0.028).RBBP8 knockdown induced DNA damage and had a synergistic effect with PARP inhibitor treatment on cell viability inhibition and cell apoptosis in AGS(generic code for human gastric adenocarcinoma cells)(t=11.154,p<0.001)and N87(t=6.362,p<0.001)cells.RBBP8 knockdown inhibited RAD51 activation and DNA terminal excision in homologous recombination repair.Conclusion:RBBP8 is involved in homologous recombination repair,and molecular intervention into RBBP8 could achieve a synthetic lethal effect with PARP inhibitor treatment in gastric cancer cells.
文摘Ovarian cancer is the most lethal gynecologic cancer. Optimal cytoreductive surgery followed by platinum-based chemotherapy with or without bevacizumab is the conventional therapeutic strategy. Since 2016, the pharmacological treatment of epithelial ovarian cancer has significantly changed following the introduction of the poly (ADP-ribose) polymerase inhibitors (PARPi). BRCA1/2 mutations and homologous recombination deficiency (HRD) have been established as predictive biomarkers of the benefit from platinum-based chemotherapy and PARPi. While in the absence of HRD (the so-called homologous recombination proficiency, HRp), patients derive minimal benefit from PARPi, the use of the antiangiogenic agent bevacizumab in first line did not result in different efficacy according to the presence of homologous recombination repair (HRR) genes mutations. No clinical trials have currently compared PARPi and bevacizumab as maintenance therapy in the HRp population. Different strategies are under investigation to overcome primary and acquired resistance to PARPi and to increase the sensitivity of HRp tumors to these agents. These tumors are characterized by frequent amplifications of Cyclin E and MYC, resulting in high replication stress. Different agents targeting DNA replication stress, such as ATR, WEE1 and CHK1 inhibitors, are currently being explored in preclinical models and clinical trials and have shown promising preliminary signs of activity. In this review, we will summarize the available evidence on the activity of PARPi in HRp tumors and the ongoing research to develop new treatment options in this hard-to-treat population.
基金the State High Technology Program "863", the National Natural Science Foundation of China (Grant No. 39880019) and Shanghai Scientific Phosphor Program.
文摘Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.
文摘Divalent metal transporter 1(DMT1)is a ferrous iron import protein.The improper expression of DMT1 is involved in neurodegenerative diseases.In the present study,we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element(DMT1-IRE)and investigated its expression and function in the C6 glioma cell line.The DMT1-IRE gene,obtained by RT-PCR,was cloned into the shuttle plasmid-ing pAdTrack-CMV containing greenfluorescent protein(GFP)reporter gene.Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escher-ichia coli(E.coli)BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I.Pac I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells(HEK293 cells),in which recombinant adenoviruses were generated within 7 to 10 days.The results demon-strated that we obtained the DMT1-IRE gene.pAdEasy1-DMT1-IRE yielded a large fragment,plus a smaller fragment of 4.5 kb after digestion with Pac I.PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE,indicating the successful construction of recombi-nant adenovirus plasmid containing DMT1-IRE.GFPfluorescence further confirmed the generation of recombi-nant AdDMT1-IRE adenovirus.AdDMT1-IRE could efficiently infect C6 glioma cells.And cell viability decreased in AdDMT1-IRE infected cells after iron overload compared to the control.These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.
文摘Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.
基金This work was partially supported by the National Key Research and Development Program of China(2016YFC0901503,2018YFA0107801)the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences,CIFMS(2017-I2M-3-015)+1 种基金the National Natural Science Foundation of China(81500156,81170470,81970149)and Tianjin Natural Science Foundation Project(20JCYBJC00470).
文摘Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further investigated the potential association between DNAH2 and the homologous recombination repair pathway of FA.The assays of homologous recombination repair,mitomycin C(MMC)sensitivity,immunofluorescence,and ubiquitination modification were performed in U2OS and DR-U2OS cell lines.In MMC-treated U2OS cells,the downregulation of the DNAH2 gene increased the sensitivity of cells to DNA inter-strand crosslinks.We also observed the reduced enrichment of FANCD2 protein to DNA damage sites.Furthermore,the ubiquitination modification level of FANCD2 was influenced by the deficiency of DNAH2.Thus,our results suggest that DNAH2 may modulate the cell homologous recombination repair partially by increasing the ubiquitination and the enrichment to DNA damage sites of FANCD2.DNAH2 may act as a novel co-pathogenic gene of FA patients.
基金Science and Technology Innovation Cultivation Fund of Zhongnan Hospital of Wuhan University,Grant/Award Number:CXPY202202Excellent Doctor Program of Zhongnan Hospital of Wuhan University,Grant/Award Number:ZNYB2021009。
文摘Background:Few studies have investigated the characteristics of non‐BRCA homologous recombination repair(HRR)pathway somatic mutations,and the impact of these mutations on efficacy of treatment in ovarian cancer patients is not clear.Therefore,we conducted this study to analyze the frequency and spectrum of somatic mutations in HRR pathway genes in patients with ovarian cancer and to examine the relationships between somatic mutations in HRR pathway genes and their effects on the efficacy of platinum‐based chemotherapy.Methods:We performed targeted sequencing of 688 genes related to the occurrence,development,treatment,and prognosis of solid tumors.Somatic mutations were identified by paired analysis of tumor tissue and germline DNA in blood cells.Results:A total of 38 patients with ovarian cancer were included in the study,and 35(92.1%)patients were diagnosed with high‐grade serous carcinoma.All patients exhibited somatic mutations in the tumor tissue samples.The commonly mutated genes were TP53(73.7%),BRCA2(55.3%),NF1(52.6%),BRCA1(47.4%),and CDH1(47.4%).Overall,71.1%of the patients exhibited mutation in at least one HRR pathway gene.The most frequently altered HRR genes were BRCA2(55.3%),followed by BRCA1(47.4%),ATM(44.7%),BARD1(42.1%),and CHEK1(36.8%).The median progression‐free survival(PFS)in patients with HRR pathway mutation was 36.0 months compared with 13.6 months in patients with no HRR pathway mutation(hazard ratio[HR],0.25;95%confidence interval[CI],0.08–0.77;p=0.016).Patients harboring BRCA1/2 and/or CDK12 mutations displayed a longer PFS(median,36.0 months)compared with patients with no BRCA1/2 or CDK12 mutation(median,13.6 months;HR,0.21;95%CI,0.07–0.61;p=0.004).In multivariate analysis Cox proportional hazards models,after adjustment for tumor stage at diagnosis and histology of initial diagnosis,patients with HRR pathway mutation had a longer PFS than patients with HRR wild‐type genes(p=0.006).Conclusions:HRR pathway somatic mutations are common in Chinese patients with ovarian cancer.HRR pathway somatic mutations were associated with improved sensitivity to platinum-based chemotherapy.Large-scale prospective studies are needed to verify our findings.
基金supported by grants from the National Key R&D Program of China(2019YFA0904000)the National Natural Science Foundation of China(31570094 and 81502962)+7 种基金the 111 Project(B16030)the Science and Technology Development Program of Suzhou(SYG201507)the Natural Science Foundation of Jiangsu Province(BK20160368)the Key Programs of Frontier Scientific Research of the Chinese Academy of Sciences(QYZDY-SSW-SMC008)the State Key Laboratory of Microbial Technology Open Projects Fund(M2017-05)the Shandong Provincial Natural Science Foundation of China(ZR2020MC015)to R.L.the Huxiang Youth Excellent(2017RS3029)to J.Y.the Taishan Scholar Program of Shandong Province to J.F.
文摘Phage-encoded homologous recombination(PEHR)is an efficient tool for bacterial genome editing.We previously developed and utilized a Pseudomonas-specific PEHR system.However,when using the PEHR system for Pseu-domonas genome editing,false positives can be a problem.In this study,we combined a compact Cascade-Cas3 system from P.aeruginosa(PaeCas3c)with a Pseudomonas-specific PEHR system,and the results of our recom-bineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy.
基金supported by the Us National Science Foundation (Awards Nos.1759831 and 2129823).
文摘Whether prokaryotes,and other microorganisms,form distinct clusters that can be recognized as species remains an issue of paramount theoretical as well as practical consequence in identifying,regulating,and communicating about these organisms.In the past decade,comparisons of thousands of genomes of isolates and hundreds of metagenomes have shown that prokaryotic diversity may be predominantly organized in such sequence-discrete clusters,albeit organisms of intermediate relatedness between the identified clusters are also frequently found.Accumulating evidence suggests,however,that the latter“intermediate”organisms show enough ecological and/or functional distinctiveness to be considered different species.Notably,the area of discontinuity between clusters often—but not always—appears to be around 85%–95%genome-average nucleotide identity,consistently among different taxa.More recent studies have revealed remarkably similar diversity patterns for viruses and microbial eukaryotes as well.This high consistency across taxa implies a specific mechanistic process that underlies the maintenance of the clusters.The underlying mechanism may be a substantial reduction in the efficiency of homologous recombination,which mediates(successful)horizontal gene transfer,around 95%nucleotide identity.Deviations from the 95%threshold(e.g.,species showing lower intraspecies diversity)may be caused by ecological differentiation that imposes barriers to otherwise frequent gene transfer.While this hypothesis that clusters are driven by ecological differentiation coupled to recombination frequency(i.e.,higher recombination within vs.between groups)is appealing,the supporting evidence remains anecdotal.The data needed to rigorously test the hypothesis toward advancing the species concept are also outlined.
基金supported by the National Key Research and Development Program of China(No.2020YFA0906800)the National Natural Science Foundation of China(Nos.31970546 and 31670061 to Y.S.,31900055 to Q.H.,31970119 to J.N.,and 31771380 to Q.S.)the State Key Laboratory of Microbial Technology.
文摘Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primarily involved in N^(6)-threonylcarbamoyl adenosine(t^(6)A)modification of transfer RNAs(tRNAs).Recently,it was reported that KEOPS participates in homologous recombination(HR)repair in yeast.To characterize the KEOPS in archaea(aKEOPS),we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus.We show that aKEOPS also possesses five subunits,Pcc1,Kae1,Bud32,Cgi121,and Pcc1-like(or Gon7-like),just like eukaryotic KEOPS.Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex(Kae1-Pcc1-Pcc1-Kae1),suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit.Strikingly,none of the genes encoding aKEOPS subunits,including Pcc1 and Pcc1-like,can be deleted in the wild type and in a t^(6)A modification complementary strain named TsaKI,implying that the aKEOPS complex is essential for an additional cellular process in this archaeon.Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI.These results suggest that aKEOPS plays an essential role independent of the cellular t^(6)A modification level.In addition,archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹCCA tail binding module.Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7.The study also reveals a possible link between the function in t^(6)A modification and the additional function,presumably HR.
基金funded by the National Key R&D Program of China(2019YFC0312504)the National Natural Science Foundation of China(41976105,81991522)+5 种基金Major Basic Research Programs of Natural Science Foundation of Shandong Province(ZR2019ZD18)the Fundamental Research Funds for the Central Universities(201941001)the NSFC-Shandong Joint Fund(U1906212)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018SDKJ0401-2)the Youth Innovation Plan of Shandong province(2019KJM004)the Taishan Scholar Youth Expert Program in Shandong Province(tsqn201812021).
文摘Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic,cephalosporin.In light of their significant medical value,an efficient gene disruption method is required for the physiological and biochemical studies on this genus of fungi.However,the number of selection markers that can be used for gene targeting is limited,which constrain the genetic analysis of multiple functional genes.In this study,we established a uridine auxotrophy based marker recycling system which achieves scarless gene deletion,and allows the use of the same selection marker in successive transformations in a deep seaderived fungus Acremonium sp.HDN16-126.We identified one homologue of Acremonium chrysogenum pyrG(also as a homologous gene of the yeast URA3)from HDN16-126,designated as pyrG-A1,which can be used as a selection marker on uridine free medium.We then removed pyrG-A1 from HDN16-126 genome via homologous recombination(HR)on MM medium with 5-fluoroortic acid(5-FOA),a chemical that can be converted into a toxin of 5-flurouracil by pyrG-A1 activity,thus generating the HDN16-126-△pyrG mutant strain which showed auxotrophy for uridine but insensitivity to 5-FOA and enabled the use of exogenous pyrG gene as both positive and negative selection marker to achieve the scarless deletion of target DNA fragments.We further applied this marker recycling system to successfully disrupt two target genes pepL(encodes a putative 2OG-Fe(Ⅱ)dioxygenase)and pepM(encodes a putative aldolase)identified from HDN16-126 genome,which are proposed to be functional genes related to 2-aminoisobutyric acid metabolism in fungi.This work is the first application of uridine auxotrophy based scarless gene deletion method in Acremonium species and shows promising potential in assisting sequential genetic analysis of filamentous fungi.
基金This work has been financially supported by the National Basic Research Program(973 Program)(Nos.2009CB918702,2005CB522804)the National Natural Science Foundation of China(Grant Nos.30623005,90608029 and 30771217)Chinese Academy of Sciences(KSCX1-YW-R-70).
文摘RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination.However,the specific pathway(s)in which it is involved and the underlining mechanism(s)remain poorly understood.We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5(dRecQ5)functions in vivo in homologous recombination-mediated double strand break(DSB)repair.We generated null alleles of dRecQ5 using the targeted recombination technique.The mutant animals are homozygous viable,but with growth retardation during development.The mutants are sensitive to both exogenous DSB-inducing treatment,such as gamma-irradiation,and endogenously induced double strand breaks(DSBs)by I-Sce I endonuclease.In the absence of dRecQ5,single strand annealing(SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion,or non-homologous end joining(NHEJ)when inter-chromosomal homologous sequence is unavailable.Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity(LOH)assays.Together,our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
基金supported by National Natural Science Foundation of China(81871944,81572389,81922067)Jiangsu 333 project(BRA2016517,China)+2 种基金Natural Science Foundation of Jiangsu Province(BK20190306,China)Jiangsu Province Key Medical Talents(ZDRCA2016026,China)Priority Academic Program Development of Jiangsu Higher Education Institutions and Fundamental Research Funds for the Central Universities(14380114,China)。
文摘Gastrointestinal mucositis is one of the most debilitating side effects of the chemotherapeutic agent irinotecan(CPT-11). Andrographolide, a natural bicyclic diterpenoid lactone, has been reported to possess anti-colitis activity. In this study, andrographolide treatment was found to significantly relieve CPT-11-induced colitis in tumor-bearing mice without decreasing the tumor suppression effect of CPT-11. CPT-11 causes DNA damage and the release of double-stranded DNA(ds DNA) from the intestine, leading to cyclic-GMP-AMP synthase(c GAS)-stimulator of interferon genes(STING)-mediated colitis, which was significantly decreased by andrographolide both in vivo and in vitro. Mechanistic studies revealed that andrographolide could promote homologous recombination(HR) repair and downregulate ds DNA-c GAS-STING signaling and contribute to the improvement of CPT-11-induced gastrointestinal mucositis. These results suggest that andrographolide may be a novel agent to relieve gastrointestinal mucositis caused by CPT-11.
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.
