-
Molecular and Cellular Biology Nov 2000The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide...
The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.
Topics: Alkylating Agents; Animals; CHO Cells; Cell Line; Cell Survival; Comet Assay; Cricetinae; Cross-Linking Reagents; DNA Repair; Dose-Response Relationship, Drug; Electrophoresis, Gel, Pulsed-Field; Kinetics; Mechlorethamine; Models, Genetic; Mutagenesis, Site-Directed; Recombination, Genetic; Rhodamines; Time Factors
PubMed: 11027268
DOI: 10.1128/MCB.20.21.7980-7990.2000 -
Stem Cell Research & Therapy Jan 2020Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical...
BACKGROUND
Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical practice, post-culture MSC supernatant treatment could be a more attractive alternative to autologous stem cell transplantation. In this study, we compared the regenerative properties of supernatants harvested from four newly established human adipose tissue mesenchymal stem cell lines (HATMSCs) derived from chronic wound patients or healthy donors.
METHODS
HATMSC supernatants were produced in a serum-free medium under hypoxia and their content was analyzed by a human angiogenesis antibody array. The regenerative effect of HATMSCs supernatants was investigated in an in vitro model of chronic wound, where cells originating from human skin, such as microvascular endothelial cells (HSkMEC.2), keratinocytes (HaCaT), and fibroblasts (MSU-1.1), were cultured in serum-free and oxygen-reduced conditions. The effect of supernatant treatment was evaluated using an MTT assay and light microscopy. In addition, fibroblasts and HATMSCs were labeled with PKH67 and PKH26 dye, respectively, and the effect of supernatant treatment was compared to that obtained when fibroblasts and HATMSCs were co-cultured, using flow cytometry and fluorescent microscopy.
RESULTS
A wide panel of angiogenesis-associated cytokines such as angiogenin, growth-regulated oncogene (GRO), interleukin-6 and 8 (IL-6, IL-8), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), and matrix metalloproteinase (MMP) were found in all tested HATMSCs supernatants. Moreover, supernatant treatment significantly enhanced the survival of fibroblasts, endothelial cells, and keratinocytes in our chronic wound model in vitro. Importantly, we have shown that in in vitro settings, HATMSC supernatant treatment results in superior fibroblast proliferation than in the case of co-culture with HATMSCs.
CONCLUSIONS
Our results suggest that therapy based on bioactive factors released by the immortalized atMSC into supernatant has important effect on skin-derived cell proliferation and might preclude the need for a more expensive and difficult cell therapy approach to improve chronic wound healing.
Topics: Adipose Tissue; Adult; Aged, 80 and over; Animals; Case-Control Studies; Cell- and Tissue-Based Therapy; Cells, Cultured; Chronic Disease; Disease Models, Animal; Humans; Mesenchymal Stem Cells; Mice; Mice, SCID; Transfection; Wound Healing; Young Adult
PubMed: 31964417
DOI: 10.1186/s13287-020-1558-5 -
Genetics May 2007There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone...
There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.
Topics: Amino Acid Sequence; DNA Damage; Histones; Models, Genetic; Molecular Sequence Data; Mutation; Oxidative Stress; Phosphorylation; Recombination, Genetic; Saccharomyces cerevisiae; Serine; Ultraviolet Rays; Vitamin K 3
PubMed: 17028320
DOI: 10.1534/genetics.106.063792 -
Current Issues in Molecular Biology Jul 2004Recombination is a ubiquitous genetic process which results in the exchange of DNA between two substrates. Homologous recombination occurs between DNA species with... (Review)
Review
Recombination is a ubiquitous genetic process which results in the exchange of DNA between two substrates. Homologous recombination occurs between DNA species with identical sequence whereas illegitimate recombination can occur between DNA with very little or no homology. Site-specific recombination is often used by temperate phages to stably integrate into bacterial chromosomes. Characterisation of the mechanisms of recombination in mycobacteria has mainly focussed on RecA-dependent homologous recombination and phage-directed site-specific recombination. In contrast the high frequency of illegitimate recombination in slow-growing mycobacteria has not been explained. The role of DNA repair in dormancy and infection have not yet been fully established, but early work suggests that RecA-mediated pathways are not required for virulence. All three recombination mechanisms have been utilised in developing genetic techniques for the analysis of the biology and pathogenesis of mycobacteria. A recently developed method for studying essential genes will generate further insights into the biology of these important organisms.
Topics: DNA, Recombinant; Genetic Markers; Genetic Vectors; Mycobacteriaceae; Recombination, Genetic; Transduction, Genetic
PubMed: 15119825
DOI: No ID Found -
Osteoarthritis and Cartilage Mar 2013Recombinant human type II collagen (rhCII) gels combined with autologous chondrocytes were tested as a scaffold for cartilage repair in rabbits in vivo.
OBJECTIVE
Recombinant human type II collagen (rhCII) gels combined with autologous chondrocytes were tested as a scaffold for cartilage repair in rabbits in vivo.
METHOD
Autologous chondrocytes were harvested, expanded and combined with rhCII-gel and further pre-cultivated for 2 weeks prior to transplantation into a 4 mm diameter lesion created into the rabbit's femoral trochlea (n = 8). Rabbits with similar untreated lesions (n = 7) served as a control group.
RESULTS
Six months after the transplantation the repair tissue in both groups filled the lesion site, but in the rhCII-repair the filling was more complete. Both repair groups also had high proteoglycan and type II collagen contents, except in the fibrous superficial layer. However, the integration to the adjacent cartilage was incomplete. The O'Driscoll grading showed no significant differences between the rhCII-repair and spontaneous repair, both representing lower quality than intact cartilage. In the repair tissues the collagen fibers were abnormally organized and oriented. No dramatic changes were detected in the subchondral bone structure. The repair cartilage was mechanically softer than the intact tissue. Spontaneously repaired tissue showed lower values of equilibrium and dynamic modulus than the rhCII-repair. However, the differences in the mechanical properties between all three groups were insignificant.
CONCLUSION
When rhCII was used to repair cartilage defects, the repair quality was histologically incomplete, but still the rhCII-repairs showed moderate mechanical characteristics and a slight improvement over those in spontaneous repair. Therefore, further studies using rhCII for cartilage repair with emphasis on improving integration and surface protection are required.
Topics: Animals; Cartilage, Articular; Case-Control Studies; Chondrocytes; Collagen Type II; Female; Femur; Gels; Hindlimb; Humans; Microscopy, Polarization; Proteoglycans; Rabbits; Spectroscopy, Fourier Transform Infrared; Stifle; Stress, Mechanical; Tissue Scaffolds; Treatment Outcome; Wound Healing; X-Ray Microtomography
PubMed: 23257243
DOI: 10.1016/j.joca.2012.12.004 -
Journal of Bacteriology Mar 1995DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded... (Comparative Study)
Comparative Study
DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.
Topics: Adenosine Triphosphatases; Conjugation, Genetic; DNA Helicases; DNA Repair; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Methyl Methanesulfonate; Mutagenesis, Insertional; RecQ Helicases; Recombination, Genetic; Sequence Deletion; Ultraviolet Rays
PubMed: 7868608
DOI: 10.1128/jb.177.5.1326-1335.1995 -
Current Biology : CB Sep 2018Crossovers (COs) are formed during meiosis by the repair of programmed DNA double-strand breaks (DSBs) and are required for the proper segregation of chromosomes. More...
Crossovers (COs) are formed during meiosis by the repair of programmed DNA double-strand breaks (DSBs) and are required for the proper segregation of chromosomes. More DSBs are made than COs, and the remaining DSBs are repaired as noncrossovers (NCOs). The distribution of recombination events along a chromosome occurs in a stereotyped pattern that is shaped by CO-promoting and CO-suppressing forces, collectively referred to as crossover patterning mechanisms. Chromosome inversions are structural aberrations that, when heterozygous, disrupt the recombination landscape by suppressing crossing over. In Drosophila species, the local suppression of COs by heterozygous inversions triggers an increase in crossing over on freely recombining chromosomes termed the interchromosomal (IC) effect [1, 2]. The molecular mechanism(s) by which heterozygous inversions suppress COs, whether noncrossover gene conversions (NCOGCs) are similarly affected, and what mediates the increase in COs in the rest of the genome remain open questions. By sequencing whole genomes of individual offspring from mothers containing heterozygous inversions, we show that, although COs are suppressed by inversions, NCOGCs occur throughout inversions at higher than wild-type frequencies. We confirm that CO frequency increases on the freely recombining chromosomes, yet CO interference remains intact. Intriguingly, NCOGCs do not increase in frequency on the freely recombining chromosomes and the total number of DSBs is approximately the same per genome. Together, our data show that heterozygous inversions change the recombination landscape by altering the relative proportions of COs and NCOGCs and suggest that DSB fate may be plastic until a CO assurance checkpoint has been satisfied.
Topics: Animals; Chromosome Inversion; Crossing Over, Genetic; DNA Breaks, Double-Stranded; Drosophila melanogaster; Genome, Insect; Heterozygote; Meiosis; Recombination, Genetic
PubMed: 30174188
DOI: 10.1016/j.cub.2018.07.004 -
Postepy Higieny I Medycyny... Dec 2014The article presents an current knowledge overview about the importance of oxidative stress and reduced efficiency of repair processes during the aging process of the... (Review)
Review
The article presents an current knowledge overview about the importance of oxidative stress and reduced efficiency of repair processes during the aging process of the human body. Oxidative damage to cellular macromolecules (proteins, lipids, nucleic acids), are formed under the influence of reactive oxygen species (ROS). They are the part of important mechanism which is responsible for the process of aging and the development of many diseases. The most important effects result from DNA damage, due to the mutations formation, which can lead to the development of tumors. However, a well-functioning repair systems (i.a. homologous recombination) remove the damage and prevent harmful changes in the cells. Lipid peroxidation products also cause oxidative modification of nucleic acids (and proteins). Proteins and fats also have repair systems, but much simpler than those responsible for the repair of nucleic acids. Unfortunately, with increasing age, they are more weakened, which contributes to increase numbers of cell damage, and consequently development of diseases specific to old age: cancer, neurodegenerative diseases or atherosclerosis.
Topics: Aging; DNA Damage; Humans; Lipid Peroxidation; Oxidative Stress; Reactive Oxygen Species; Wound Healing
PubMed: 25531712
DOI: 10.5604/17322693.1132010 -
PloS One 2015The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed...
The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination.
Topics: Bacteriophage lambda; DNA Repair; DNA Replication; DNA, Single-Stranded; Escherichia coli; Recombination, Genetic
PubMed: 25803509
DOI: 10.1371/journal.pone.0120681 -
Proceedings of the National Academy of... Jun 1985The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria....
The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed.
Topics: DNA Repair; Dose-Response Relationship, Radiation; Enterobacteriaceae; Mutation; Rec A Recombinases; Recombination, Genetic
PubMed: 3858873
DOI: 10.1073/pnas.82.12.4172