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1.
Two classes of homologous recombination mechanism for repair of double-strand breaks (DSBs) have been described in eukaryotes so far. One is conservative and has been explained by the double-strand break repair model (Szostak et al., 1983), whereas the other one is non-conservative and has been explained by the single-strand annealing model (Lin et al., 1984). Here, we will review data supporting the existence of another homologous recombination mechanism for double-strand break repair. We will present the one-sided invasion model that we have proposed to explain this mechanism and discuss its potential implication in various homologous recombination events. 相似文献
2.
MA Resnick 《Canadian Metallurgical Quarterly》1976,59(1):97-106
3.
We have investigated the role of DNA ends during gap repair by homologous recombination. Mouse cells were transfected with a gapped plasmid carrying distinctive ends: on one side mouse LINE-1 repetitive sequences (L1Md-A2), and on the other rat LINE-1 sequences (L1Rn-3). The gap could be repaired by homologous recombination with endogenous mouse genomic LINE-1 elements, which are on average 95% and 85% homologous to L1Md-A2 and L1Rn-3 ends, respectively. Both L1Md-A2 and L1Rn-3 ends were found to initiate gap repair with equal efficiency. However, there were two types of gap repair products--precise and imprecise--the occurrence of which appears to depend on which end had been used for initiation and thus which end was left available for subsequent steps in recombination. These results, together with sequence analysis of recombinants obtained with plasmids having either mouse or rat LINE-1 sequences flanking the gap, strongly suggest that the two DNA ends played different roles in recombinational gap repair. One end was used to initiate the gap repair process, while the other end was involved at later steps, in the resolution of the recombination event. 相似文献
4.
In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model. 相似文献
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Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination. 相似文献
7.
Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. 相似文献
8.
STATEMENT OF PROBLEM: The cone-screw abutment has been shown to diminish micromovement by reducing the burden of component loosening and fracture. However, anecdotal concern for cold welding of cone-screw joints in implant design has been identified as a potential source for lack of retrievability. PURPOSE: This comparative study evaluated the loosening torque, as a percentage of tightening torque, for the ITI Straumann and Astra Tech (3.5 and 4.0 mm diameters) implant systems, which use an 8-degree and 11-degree internal cone, respectively. MATERIAL AND METHODS: Implants and abutments from each system were mounted in a torque device, and a range of tightening torques was applied. Loosening torques were then measured, and the influence of conus angle, interfacial surface area, saliva contamination, and time delay to loosening were all assessed. RESULTS: The loosening torque only exceeded tightening torque at the highest levels, just before component failure, when plastic deformation was expected. For all clinically relevant levels of torque, both in a dry environment and with components bathed in artificial saliva at 37 degrees C, loosening torque was always seen to be 80% to 90% of tightening torque, demonstrating that cold welding does not occur. There was a high correlation between loosening and tightening torque for all systems tested, but no statistical difference when comparing wet versus dry or comparing individual data for each system. CONCLUSIONS: It can be concluded that for clinically relevant levels of tightening torque, no problems are anticipated with respect to retrievability. 相似文献
9.
Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product. 相似文献
10.
Cassette mutagenesis was used to produce a library of mutations at the interface of the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c. The library is random and comprises > 98% mutations. Over 11,000 candidates were assayed for function by selecting for the ability of yeast, with the mutated gene as their sole cytochrome c source, to grow on nonfermentable carbon sources. We estimate that approximately 0.5% of the 160,000 total amino acid combinations at these four residues result in a functional cytochrome c. Significant correlations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cyclohexane-to-water and n-octanol-to-water). Similar correlations are observed with respect to growth rate. Finally, sequences that are consistent with function follow a binary amino acid pattern. 相似文献
11.
Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase. 相似文献
12.
Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non-randomness for dsb induction is discussed. 相似文献
13.
M Wada Y Ihara M Tatsuka H Mitsui K Kohno M Kuwano D Schlessinger 《Canadian Metallurgical Quarterly》1994,200(3):1693-1700
The transfer of a yeast artificial chromosome (YAC) into mouse and human cell lines was effected by four methods, and the efficiency and integrity of the incorporated YAC DNA were compared. A 500 kb YAC containing the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was transferred more efficiently by polyethylene glycol-mediated fusion than by lipofection, electrofusion, or electroporation. Southern blot analysis demonstrated that PEG fusion lines yielded fragments of the size of the original YAC clone, whereas lipofection and electroporation did not. Two of 53 fusion lines showed 6-thioguanine resistance and confirmatory disruption of the HPRT gene in the YAC DNA, suggesting that the YAC DNA was integrated by homologous recombination with the endogenous HPRT gene region. 相似文献
14.
In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 5'-->3' exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 3'-->5' exonuclease processing also occurs. 相似文献
15.
C Venderbure A Chastanet F Boudsocq S Sommer A Bailone 《Canadian Metallurgical Quarterly》1999,181(4):1249-1255
By its functional interaction with a RecA polymer, the mutagenic UmuD'C complex possesses an antirecombination activity. We show here that MucA'B, a functional homolog of the UmuD'C complex, inhibits homologous recombination as well. In F- recipients expressing MucA'B from a Ptac promoter, Hfr x F- recombination decreased with increasing MucA'B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA'B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA'B together with UmuD'C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD'C inhibition of recombination, are inhibited by MucA'B while promoting MucA'B-promoted mutagenesis efficiently. The data suggest that MucA'B and UmuD'C contact a RecA polymer at distinct sites. The MucA'B complex was more active than UmuD'C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD'C does. The enhanced mutagenic potential of MucA'B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells. 相似文献
16.
We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E. coli strains. Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks. 相似文献
17.
Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex. 相似文献
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For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu+ transformants (micrograms pRPLeu10 DNA)-1 (1 x 10(6) viable protoplasts)-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus. 相似文献
20.
The photochemical (lambda < 400 nm) decomposition of some monocyclic and polycyclic nitramines produces .NO2, which can be detected in the respective nitramine crystals at 77 K by EPR (electron paramagnetic resonance). In solutions of perdeutero-dimethylsulfoxide (DMSO-d6) the .NO2 produced by photolytic decomposition of dissolved nitramines can be spintrapped by the solvent to give a radical having the structure CD3-(SO2)-(NO.)-CD3. In this article, we examine this reaction for two nitramines: cyclotrimethylenetrinitramine (RDX) and hexanitrohexaazaisowurzitane (HNIW), which are energetic materials. The decay of the spin-adduct radical (I) follows first-order kinetics for both nitramines studied, having a rate constant (k) of congruent to 7.1 x 10(-4) s-1. The net growth in spin concentration of (1) measured from EPR spectra is fitted by a first-order rate equation taking into account the simultaneous competitive decay rate of spin adduct (I). Using the rate data and EPR spin concentration data, the ratio of free .NO2 produced per parent nitramine molecule is estimated as 1:1 for RDX and 4:1 for HNIW. Biological implications of trapping of .NO2 by dimethyl sulfoxide are discussed. 相似文献