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Proceedings of the National Academy of... Aug 1996The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this...
The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku autoantigen, a protein that binds to DNA ends and recruits the DNA-dependent protein kinase to DNA. Using an I-SceI endonuclease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO-K1 cell lines. A DSB in chromosomal DNA increased the yield of recombinants several thousand-fold above background in both the xrs-6 and CHO-K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector. Thus, recombinational repair of chromosomal DSBs can occur at substantial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chromosome ends (end-joining) near the site of the DSB was also examined. In contrast to recombinational repair, end-joining was found to be severely impaired in the xrs-6 cells. Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways.
Topics: Animals; Antigens, Nuclear; Base Sequence; Chromosomes; Cricetinae; DNA Helicases; DNA Repair; DNA-Binding Proteins; Deoxyribonucleases, Type II Site-Specific; Gene Targeting; Ku Autoantigen; Molecular Sequence Data; Nuclear Proteins; Radiation Tolerance; Recombination, Genetic; Saccharomyces cerevisiae Proteins
PubMed: 8799130
DOI: 10.1073/pnas.93.17.8929 -
Bulletin Du Cancer Mar 2011
Topics: Cell Fusion; Chromosome Breakage; DNA Fragmentation; DNA Repair; Gene Amplification; Gene Rearrangement; Neoplasms; Polymorphism, Single Nucleotide; Recombination, Genetic; Sequence Analysis, DNA
PubMed: 21644265
DOI: No ID Found -
Molecular Cell Oct 2002Tolerance mechanisms are important in the ability of cells to cope with DNA damage. In E. coli, the two main damage tolerance mechanisms are recombinational repair (RR)...
Tolerance mechanisms are important in the ability of cells to cope with DNA damage. In E. coli, the two main damage tolerance mechanisms are recombinational repair (RR) and translesion replication (TLR). Here we show that RR effectively repairs gaps opposite DNA lesions. When both mechanisms are functional, RR predominates over TLR, being responsible for 86% of the repair events. This predominance of RR is determined by the high concentration of RecA present under SOS conditions, which causes a differential inhibition of TLR. Further inhibition of TLR is caused by the RecA-catalyzed strand exchange reaction of RR. This molecular hierarchy in the tolerance of DNA lesions ensures that the nonmutagenic RR predominates over the mutagenic TLR, thereby contributing to genetic stability.
Topics: Bacterial Proteins; DNA Damage; DNA Repair; DNA Replication; DNA, Bacterial; DNA-Directed DNA Polymerase; Escherichia coli; Escherichia coli Proteins; Mutagenesis; Rec A Recombinases; Recombination, Genetic; SOS Response, Genetics
PubMed: 12419234
DOI: 10.1016/s1097-2765(02)00679-2 -
Osteoarthritis and Cartilage Nov 2006Microfracture is used to treat articular cartilage injuries, but leads to the formation of fibrocartilage rather than native hyaline articular cartilage. Since bone...
OBJECTIVE
Microfracture is used to treat articular cartilage injuries, but leads to the formation of fibrocartilage rather than native hyaline articular cartilage. Since bone morphogenetic protein 7 (BMP-7) induces cartilage differentiation, we hypothesized that the addition of the morphogen would improve the repair tissue generated by microfracture. We determined the effects of these two treatments alone and in combination on the quality and quantity of repair tissue formed in a model of full-thickness articular cartilage injury in adolescent rabbits.
DESIGN
Full-thickness defects were made in the articular cartilage of the patellar grooves of forty, 15-week-old rabbits. Eight animals were then assigned to (1) no further treatment (control), (2) microfracture, (3) BMP-7, (4) microfracture with BMP-7 in a collagen sponge (combination treatment), and (5) microfracture with a collagen sponge. Animals were sacrificed after 24 weeks at 39 weeks of age. The extent of healing was quantitated by determining the thickness and the surface area of the repair tissue. The quality of the repair tissue was determined by grading specimens using the International Cartilage Repair Society Visual Histological Assessment Scale.
RESULTS
Compared to controls, BMP-7 alone increased the amount of repair tissue without affecting the quality of repair tissue. Microfracture improved both the quantity and surface smoothness of repair tissue. Compared to either single treatment, the combination of microfracture and BMP-7 increased both the quality and quantity of repair tissue.
CONCLUSIONS
Microfracture and BMP-7 act synergistically to stimulate cartilage repair, leading to larger amounts of repair tissue that more closely resembles native hyaline articular cartilage.
Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Collagen; Combined Modality Therapy; Disease Models, Animal; Fractures, Cartilage; Hindlimb; Male; Minimally Invasive Surgical Procedures; Rabbits; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome; Wound Healing
PubMed: 16765606
DOI: 10.1016/j.joca.2006.04.004 -
BMC Molecular Biology Mar 2014Double Stranded Breaks (DSBs) are the most serious form of DNA damage and are repaired via homologous recombination repair (HRR) or non-homologous end joining (NHEJ)....
BACKGROUND
Double Stranded Breaks (DSBs) are the most serious form of DNA damage and are repaired via homologous recombination repair (HRR) or non-homologous end joining (NHEJ). NHEJ predominates in mammalian cells at most stages of the cell cycle, and it is viewed as 'error-prone', although this notion has not been sufficiently challenged due to shortcomings of many current systems. Multi-copy episomes provide a large pool of genetic material where repair can be studied, as repaired plasmids can be back-cloned into bacteria and characterized for sequence alterations. Here, we used EBV-based episomes carrying 3 resistance marker genes in repair studies where a single DSB is generated with virally-encoded HO endonuclease cleaving rapidly at high efficiency for a brief time post-infection. We employed PCR and Southern blot to follow the kinetics of repair and formation of processing intermediates, and replica plating to screen for plasmids with altered joints resulting in loss of chloramphenicol resistance. Further, we employed this system to study the role of Metnase. Metnase is only found in humans and primates and is a key component of the NHEJ pathway, but its function is not fully characterized in intact cells.
RESULTS
We found that repair of episomes by end-joining was highly accurate in 293 T cells that lack Metnase. Less than 10% of the rescued plasmids showed deletions. Instead, HEK293 cells (that do express Metnase) or 293 T transfected with Metnase revealed a large number of rescued plasmids with altered repaired joint, typically in the form of large deletions. Moreover, quantitative PCR and Southern blotting revealed less accurately repaired plasmids in Metnase expressing cells.
CONCLUSIONS
Our careful re-examination of fidelity of NHEJ repair in mammalian cells carrying a 3' cohesive overhang at the ends revealed that the repair is efficient and highly accurate, and predominant over HRR. However, the background of the cells is important in establishing accuracy; with human cells perhaps surprisingly much more prone to generate deletions at the repaired junctions, if/when Metnase is abundantly expressed.
Topics: Cell Line; DNA Breaks, Double-Stranded; DNA Damage; DNA End-Joining Repair; HEK293 Cells; Herpesvirus 4, Human; Histone-Lysine N-Methyltransferase; Humans; Plasmids; Recombination, Genetic
PubMed: 24655462
DOI: 10.1186/1471-2199-15-6 -
Molecular Medicine Reports Sep 2020Local transplantation of epidermal stem cells (ESCs) exerts a therapeutic effect on burn wounds. However, cell viability can impede their clinical application. HOX...
Local transplantation of epidermal stem cells (ESCs) exerts a therapeutic effect on burn wounds. However, cell viability can impede their clinical application. HOX antisense intergenic RNA (HOTAIR) is involved in regulating adult tissue stem cells, as well as in developmental patterning and pluripotency. However, little is known about its role in regulating ESCs. The present study was performed to investigate the effects of HOTAIR in the modulation of ESCs and wound repair. Firstly, reverse transcription‑quantitative PCR was used to detect the relative expression of HOTAIR during burn wound healing in mice to determine whether HOTAIR is associated with wound healing. Subsequently, ESCs derived from mouse skin were transfected with a lentiviral vector to overexpress or knockdown HOTAIR. The effects of HOTAIR on cell proliferation and differentiation were measured by 5‑bromodeoxyuridine and MTT assays, and by assessing NANOG mRNA expression. Lastly, mice with burns were administered a subcutaneous injection of HOTAIR‑overexpressing ESCs. Images were captured and histological analyses were performed to evaluate wound healing. The results revealed that the expression of HOTAIR gradually increased and peaked at day 7 post‑burn and maintained at relatively high levels until day 14 post‑burn during wound healing. Furthermore, overexpression of HOTAIR promoted ESC proliferation and maintained the stem cell state in vitro. By contrast, suppression of HOTAIR inhibited cell proliferation and cell stemness. It was also identified that HOTIR‑overexpressing ESCs accelerated re‑epithelialization and facilitated burn wound repair. In conclusion, the present findings confirmed an essential role of HOTAIR in the regulation of ESC proliferation and stemness. Therefore, targeting HOTAIR in ESCs may be a potentially promising therapy for burn wound healing.
Topics: Animals; Burns; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epidermal Cells; Female; Injections, Subcutaneous; Mice; Nanog Homeobox Protein; RNA, Long Noncoding; Stem Cell Transplantation; Stem Cells; Transfection; Wound Healing
PubMed: 32582996
DOI: 10.3892/mmr.2020.11268 -
Molecular and Cellular Biology Nov 2004Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate...
Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast.
Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.
Topics: Base Sequence; Carrier Proteins; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosomes, Fungal; Cloning, Molecular; DNA Damage; DNA Repair; DNA Replication; DNA, Fungal; DNA-Binding Proteins; Epistasis, Genetic; Genes, Essential; Genes, Fungal; Molecular Sequence Data; Multiprotein Complexes; Mutation; Protein Binding; Rad51 Recombinase; Recombination, Genetic; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Sequence Alignment
PubMed: 15485909
DOI: 10.1128/MCB.24.21.9401-9413.2004 -
Molecular Microbiology Nov 2017RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a...
RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF-dr1088 operon, which is highly conserved in the Deinococcus-Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two-hybrid and pull-down assays. DR1088 exhibited some RecO-like biochemical properties including single/double-stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity. However, unlike other recombination proteins, dr1088 is essential for cell viability. These results indicate that DR1088 might play a role in DNA replication and DNA repair processes.
Topics: Bacterial Proteins; DNA Repair; DNA Replication; DNA, Bacterial; DNA, Single-Stranded; DNA-Binding Proteins; Deinococcus; Rec A Recombinases; Recombination, Genetic; Recombinational DNA Repair
PubMed: 28862774
DOI: 10.1111/mmi.13828 -
Proceedings of the National Academy of... Jul 2001Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes...
Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by gamma-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.
Topics: Cell Nucleus; DNA Damage; DNA Repair; DNA, Fungal; DNA-Binding Proteins; Deoxyribonucleases, Type II Site-Specific; Fungal Proteins; Intracellular Signaling Peptides and Proteins; Mitosis; Protein Serine-Threonine Kinases; Rad52 DNA Repair and Recombination Protein; Recombinant Fusion Proteins; Recombination, Genetic; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 11459964
DOI: 10.1073/pnas.121006298 -
Nucleic Acids Research Mar 1988We have shown previously that heteroduplexes containing single-stranded loops are repaired efficiently in monkey cells, but not always correctly: 2% of the repair...
We have shown previously that heteroduplexes containing single-stranded loops are repaired efficiently in monkey cells, but not always correctly: 2% of the repair products acquired mutations within a 350 base-pair target (Weiss, U. and Wilson, J.H., Proc. Natl. Acad. Sci. USA 87:1123-1126, 1987). The structures of the mutant genomes, which are described here, are consistent with an error-prone repair system. The spectrum of mutations includes about 25% point mutations and 75% rearrangements, which consist of deletions, duplications, and substitutions. The mutations are clustered in the vicinity of single-stranded loops in the original heteroduplex. The high frequency of mutation, their clustering, and the positions of rearrangement endpoints suggest that the mutations were generated during repair of the heteroduplexes.
Topics: Animals; Base Sequence; Cell Line; Chlorocebus aethiops; DNA Mutational Analysis; DNA Repair; DNA, Viral; Recombination, Genetic; Simian virus 40; Transfection
PubMed: 2833731
DOI: 10.1093/nar/16.5.2313