Neutrophil depletion retards endometrial repair in a mouse model

The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair. This work was funded by the National Health and Medical Research Council of Australia (#143798, #241000) and by an Australian Postgraduate Scholarship to T.K.     

Polymorphisms in the human XPD (ERCC2) gene, DNA repair capacity and cancer susceptibility: an appraisal

Using the human XPD (ERCC2) gene as an example, we evaluate the suggestion that polymorphisms in DNA repair genes lead to decreased DNA repair capacity and to increased cancer susceptibility. This intuitively appealing idea provides the rationale for a large number of studies that have attracted much attention from scientists, clinicians and the general public. Unfortunately, most of this work presupposes that a functional effect has been established for the DNA repair gene polymorphisms under study. For XPD, there is no credible evidence for any effect on DNA repair of the two common polymorphisms leading to p.D312N and p.K751Q amino acid variations, and evolutionary analyses strongly predict that both polymorphisms are benign. Current evidence suggests no causal relationship between XPD polymorphisms, reduced DNA repair and increased cancer risk. We do not believe that more studies of the same kind will be useful. Instead, we suggest a combination of several other approaches, which up to now have been used in only a sporadic way, to examine more rigorously the possibility that phenotypic differences are associated with polymorphisms in other DNA repair genes.     

Escherichia coli as a model system to study DNA repair genes of eukaryotic organisms

The bacteria Escherichia coli has been widely employed in studies of eukaryotic DNA repair genes. Several eukaryotic genes have been cloned by functional complementation of mutant lineages of E. coli. We examined the similarities and differences among bacterial and eukaryotic DNA repair systems. Based on these data, we examined tools used for gene cloning and functional studies of DNA repair in eukaryotes, using this bacterial system as a model.     

Functional validation of miRNAs targeting genes of DNA double-strand break repair to radiosensitize non-small lung cancer cells

DNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway.     

Marker structure and recombination substrate environment influence conversion preference of broken and unbroken alleles in Saccharomyces cerevisiae

Double-strand break (DSB)-induced gene conversion was investigated using plasmid x chromosome (P x C) and chromosomal direct-repeat recombination substrates with markers arranged such that functional (selected) products could not arise by longpatch mismatch repair initiated from the DSB. As seen previously with analogous substrates, these substrates yield products with discontinuous conversion tracts, albeit at low frequency. Most conversion tracts were of minimum length, suggesting that heteroduplex DNA (hDNA) is limiting, or that co-repair imposes selective pressure against products with more extensive hDNA. When functional products can arise by long-patch mismatch repair, the broken allele is converted in nearly all products. In contrast, in the absence of long-patch mismatch repair, unbroken alleles are frequently converted, and we show that such conversion depends on both marker structure (i.e., long palindromic vs. nonpalindromic insertions) and the chromosomal environment of the recombination substrate. We propose that conversion of unbroken alleles is largely a consequence of the segregation of unrepaired markers, and that differences in mismatch repair efficiency underlie the observed effects of marker structure and chromosome environment on allele conversion preference.     

A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae

The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.     

Nuclear-gene targeting by using single-stranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii

Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3'-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.     

Saccharomyces cerevisiae DNA repair processes: an update

The budding yeast Saccharomyces cerevisiae plays a central role in contributing to the understanding of one of the most important biological process, DNA repair, that maintains genuine copies of the cellular chromosomes. DNA lesions produce either spontaneously or by DNA damaging agents are efficiently repaired by one or more DNA repair proteins. While some DNA repair proteins function independently as in the case of base excision repair, others belong into three separate DNA repair pathways, nucleotide excision, mismatch, and recombinational. Of these pathways, nucleotide excision and mismatch repair show the greatest functional conservation between yeast and human cells. Because of this high degree of conservation, yeast has been regarded as one of the best model system to study DNA repair. This report therefore updates current knowledge of the major yeast DNA repair processes.     

Dominant sensitization variants of human O(6)-methylguanine-DNA-methyltransferase obtained by a mutational screen of surface residues

A scanning mutagenesis experiment was performed on human O(6)-methylguanine methyltransferase (hMGMT), directed largely at non-conserved surface residues that have not previously been studied. Variants typically contained two or more substitutions. Two of the 16 variants characterized in detail are inactive for methyltransfer, but increase the cytotoxicity and mutagenic effects of methylating agents. This phenotype is reminiscent of a variant (C145A) that has a mutation in the methyl-accepting cysteine. C145A is inactive, but reportedly binds methylated DNA and confers sensitivity to methylating agents. The sensitization phenotype of the two new variants is more striking in strains that are wild-type for DNA repair than in strains that are deficient for repair, suggesting that these proteins inhibit functional DNA repair proteins by competitively binding to methylated DNA. Both variants have multiple substitutions in the last helix of the protein. These results suggest that the C-terminal helix is necessary for methyltransfer activity, but not for methylguanine-specific binding.     

Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.     

Differential repair of excision gaps generated by transposable elements of the ‘Ac family’

Studies on transposable elements of the Ac family have led to different models for excision gap repair in either plants or Drosophila. Excision products generated by the plant transposable elements Ac and Tam3 imply a more or less straightforward ligation of broken ends; excision products of the Drosophila P element indicate the involvement of ‘double-strand break’ (DSB) repair. Recent findings that excision products of Ac and Tam3 can also contain traces of the element ends indicate, however, that DSB repair might be an alternative repair mechanism in plants. A functional DSB repair mechanism in plants can also be deduced from the observed rapid increases of Ac copy number during plant development and from the involvement of Ac in the generation of internal Ac deletions. On the other hand, alternative repair mechanisms may also be functional in Drosophila, because some of the ‘footprints’ generated upon P excision can be explained by a mechanism that has been postulated for excision gap repair in plants. It is concluded that plants and Drosophila can use similar repair mechanisms, but that the predominance of a certain repair mechanism is determined by the host.     

The genetics and specificity of the constitutive excision repair system of Bacillus subtilis

An isogenic set of DNA repair-proficient and -deficient strains of B. subtilis, cured of all prophages, were constructed and analyzed for their sensitivities to selected mutagens. The results demonstrated that the lethal damage caused by ultraviolet (UV) radiation and by 4-nitroquinoline-1-oxide (4NQO) were repaired by the bacterial excision and/or recombination repair systems. In contrast, the lethal damages caused by ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS) were removed from the DNA by the recombination repair system of the bacteria, and not by the excision repair system. Significantly, the bacteria required both a functional recombination repair system and a functional excision repair system in order to remove the DNA damage caused by the bifunctional alkylating agent mitomycin C (MC).     

From Inflammation to Wound Healing: Using a Simple Model to Understand the Functional Versatility of Murine Macrophages

Macrophages are fundamental cells of the innate immune system. Their activation is essential for such distinct immune functions as inflammation (pathogen-killing) and tissue repair (wound healing). An open question has been the functional stability of an individual macrophage cell: whether it can change its functional profile between different immune responses such as between the repair pathway and the inflammatory pathway. We studied this question theoretically by constructing a rate equation model for the key substrate, enzymes and products of the pathways; we then tested the model experimentally. Both our model and experiments show that individual macrophages can switch from the repair pathway to the inflammation pathway but that the reverse switch does not occur.     

Functional analysis of Drosophila melanogaster BRCA2 in DNA repair

The human BRCA2 cancer susceptibility protein functions in double-strand DNA break repair by homologous recombination and this pathway is conserved in the fly Drosophila melanogaster. Although a potential Drosophila melanogaster BRCA2 orthologue (dmbrca2; CG30169) has been identified by sequence similarity, no functional data addressing the role of this protein in DNA repair is available. Here, we demonstrate that depletion of dmbrca2 from Drosophila cells induces sensitivity to DNA damage induced by irradiation or treatment with hydroxyurea. Dmbrca2 physically interacts with dmrad51 (spnA) and the two proteins become recruited to nuclear foci after DNA damage. A functional assay for DNA repair demonstrated that in flies dmbrca2 plays a role in double-strand break repair by gene conversion. Finally, we show that depletion of dmbrca2 in cells is synthetically lethal with deficiency in other DNA repair proteins including dmparp. The conservation of the function of BRCA2 in Drosophila will allow the analysis of this key DNA repair protein in a genetically tractable organism potentially illuminating mechanisms of carcinogenesis and aiding the development of therapeutic agents.     

Phenotypic plasticity and effects of selection on cell division symmetry in Escherichia coli

Aging has been demonstrated in unicellular organisms and is presumably due to asymmetric distribution of damaged proteins and other components during cell division. Whether the asymmetry-induced aging is inevitable or an adaptive and adaptable response is debated. Although asymmetric division leads to aging and death of some cells, it increases the effective growth rate of the population as shown by theoretical and empirical studies. Mathematical models predict on the other hand, that if the cells divide symmetrically, cellular aging may be delayed or absent, growth rate will be reduced but growth yield will increase at optimum repair rates. Therefore in nutritionally dilute (oligotrophic) environments, where growth yield may be more critical for survival, symmetric division may get selected. These predictions have not been empirically tested so far. We report here that Escherichia coli grown in oligotrophic environments had greater morphological and functional symmetry in cell division. Both phenotypic plasticity and genetic selection appeared to shape cell division time asymmetry but plasticity was lost on prolonged selection. Lineages selected on high nutrient concentration showed greater frequency of presumably old or dead cells. Further, there was a negative correlation between cell division time asymmetry and growth yield but there was no significant correlation between asymmetry and growth rate. The results suggest that cellular aging driven by asymmetric division may not be hardwired but shows substantial plasticity as well as evolvability in response to the nutritional environment.     

Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination

The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.     

A multivariate approach to the treatment of peripheral nerve transection injury: the role of electromagnetic field therapy

A multivariate approach to the treatment of peripheral nerve transection injury has been used in a rat model. A pilot study (48 animals, 8 groups) examined variables associated with the method and timing of surgical repair, the arrest of wallerian degeneration, and the role of pulsing electromagnetic field therapy (PEMF) in functional recovery. A second phase (90 animals, 6 groups) then studied the timing and duration of pulsing electromagnetic field therapy as the only variable in larger groups of animals. The pilot study revealed that a vein-graft conduit did not improve functional recovery compared with standard epineurial repair. Additionally, delayed repair compared favorably with immediate repair. The use of chlorpromazine to inhibit the toxic effects of calcium influx appeared to enhance early functional recovery, and the combination of delayed nerve repair and pulsing electromagnetic field therapy seemed to consistently improve function. The second phase of the study has demonstrated (for the first time) statistical improvement in ambulation in animals treated with delayed surgical repair and prolonged pulsing electromagnetic field therapy. We postulate that future treatment of nerve transection injuries will involve a combined treatment regimen consisting of the immediate arrest of wallerian degeneration, delayed surgery, and pulsing electromagnetic field therapy.