Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study inDietziasp. Strain DQ12-45-1b

ABSTRACTNonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo. Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, anEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms.IMPORTANCEMany nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.

Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

Non Homologous End Joining, a Marker Of Genomic Instability Is Elevated In Multiple Myeloma: A New Prognostic Factor

Genomic instability is a hallmark of several types of solid and hematologic malignancies, including multiple myeloma (MM). Although structural and numerical chromosomal abnormalities are common features of MM cells, the underlying molecular basis of MM genomic instability is still largely unknown. To this aim, we have investigated the activity of non-homologous end joining (NHEJ), which represents the most important mechanism of double-strand breaks (DSBs) repair, in MM cells. First, we developed and validated a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene which measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency, in either intact cells (in vivo assay) or in cell free extracts (in vitro assay). The first one is a chemiluminescent assay which allows for direct measurement of LUC and SEAP in the supernatant of the cells 24h after electroporation with the plasmid, while the cell free extract method is a customized TaqMan® approach based on a quantitative evaluation of the plasmid rejoining. Both assays revealed a significant increase in NHEJ in all 6 MM cell lines tested compared to normal peripheral blood mononuclear cells (PB-ND) and bone marrow stromal cells (BMSC). We further confirmed the hyper-activation of the NEHJ pathway by analyzing the binding activity of ku86, a key NHEJ-related protein involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. Six out of 9 MM cell lines showed a significant increase in ku86-binding activity respect to normal cells. We also found a higher phosphorylation at Ser 2056 of DNA-PK, a ku86-partner whitch plays a key role in NHEJ. Next, we evaluated the NHEJ activity in 35 patient samples using the cell free assay. Interestingly, level of NHEJ activity divided patients into two different groups: one with an NHEJ activity similar to normal cells and the other to the MM cell lines. Preliminary correlation analysis between NHEJ activity and the clinical features of the patients indicated that the MGUS and Smoldering MM subgroup fall into the normal cluster while relapsed/refractory disease to the cell lines one. Finally, using the French (IFM) and the Arkansas (GSE2658) dataset, we demonstrate a significant association between NHEJ pathway-related gene expression and overall survival by the Globaltest analysis. We were also able to find a common NHEJ signature of 6 genes whose expression significantly correlates with patient survival in both the datasets. In conclusion, our data indicate an aberrant activation of NHEJ in MM, highlighting its role in the progression of the disease and suggesting this pathway as an important new prognostic marker in myeloma. Disclosures: No relevant conflicts of interest to declare.

Study of Expression of Ku70 and NHEJ in CML Cells

Objective FThe DNA double strand breaks (DSB) in mammalian cells are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). Ku70 played a pivotal role in NHEJ pathway. As a common hematological malignant disease with unique chromosomal translocation t (9;22), chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As we reported before, protein Ku70 expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in blast phase was markedly higher than that in chronic phase. The present study furthermore aims to investigate the expression of the gene Ku70 in CML cells at different clinical stage and reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR-ABL in cells of CML. The NHEJ efficiency to repair DSB in CML was also investigated. Methods: Bone marrow cells were collected from 24 cases of normal adults and 27 cases of de novo diagnosed CML patients. 15 CML patients were in chronic phase and 12 in blast phase. The expression of gene Ku70 was detected by RT-PCR and RQ-RT-PCR. The fusion gene BCR-ABL was detected by TaqMan probe Real Time PCR, using ABL as the internal control. Nucleic extracted proteins were used to determine NHEJ efficiency by an in vitro end-ligation system. Results: The mean level of NHEJ activity of normal BM cells and CML cells were 18.6±13.1% vs 24.8±14.9%, .024. The gene Ku 70 expression in normal BM cells and CML cells were 31.08±8.41 vs 544.63±1185.71 copies/10,000 β-Actin copies, P=0.039. gene Ku 70 expressed significantly higher in blast phase (1103.31±1645.62 copies/10,000 β-Actin copies, P<0.01). There were positive correlations of BCR-ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) and with the protein Ku70 (r=0.705 P<0.001) in CML cells. There was also significantly correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P<0.001). Conclusions: The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells with NHEJ activity was also enhanced in CML cells. Meanwhile, Ku70 expression level in acute phase was markedly higher than that in chronic phase. There were significantly correlations among the expression of fusion gene BCR-ABL, the gene Ku70 and the protein Ku70 in cells of CML This Study illustrates DNA instability in CML cells which was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. NHEJ pathway plays an important role in disease progression of CML.