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− | + | Nucleotides are shown in blue. The F chain of EcHda and also the A chain of SaHda are displayed inside the identical orientation. (B) The superimposed structures of EcHda (chain F, teal) from the Hda?clamp complex and SaHda (3BOS; chain A, orange) collectively with secondary structural elements are shown. ADP in EcHda and CDP in SaHda are shown in magenta and blue sticks, respectively. Each N-terminus of EcHda or SaHda is marked in the bottom with red or blue dots, respectively. (C) Close-up view from the nucleotide-binding web site of the superimposed structures shows the interactions between nucleotide and hydrophobic residues with the lid domain. (D) The superimposed ADP and CDP of EcHda and SaHda, as well as the -phosphate and -phosphate marked by yellow or cyan dots are shown. The sticks are colored as in (B).sensitivity reflects a defect in the interactions among Hda protomers, and/or amongst Hda and DnaA. We also measured the cellular DNA content on the mutant hda strains applying flow cytometry (Figure 6B). The wildtype strain showed big peaks at 2n and 4n positions, as did the hda-Q142A, hda-N144A and hdahook mutants. In contrast, the hda-E126A, hda-E126A-K81R, hda-W156A and hda-Y160A mutants exhibited a single broad peakgreater than 4n (Figure 6B), indicating asynchronous initiation that is definitely consistent with a RIDA defect. We also measured oriC:TerC ratios in these strains (Figure 6C). Compared with the wild-type handle, the hda diaA strains displayed an expected significantly improved oriC:TerC ratio, indicating an excessive frequency of initiations. Whereas the hda-E126A mutant was also elevated, consistent having a RIDA defect, the other hda mutants [https://britishrestaurantawards.org/members/singer20europe/activity/356469/ https://britishrestaurantawards.org/members/singer20europe/activity/356469/] integrated the hda-3900 Nucleic Acids Analysis, 2017, Vol. 45, No.Figure six. In vivo and In vitro analyses of Hda mutants (A) In vivo test of sensitivity of Escherichia coli mutants to hydroxyurea. Just after serial dilution, the sensitivity of E. coli strains encoding mutant Hdas to the indicated concentrations of HU was measured as described in `Materials and Methods' section. Cultures had been normalized for cell density depending on OD600nm before serial dilutions. (B) Flow cytometry evaluation of strains expressing hda alleles. Cells (50 000) in the indicated strains had been analyzed immediately after staining with PicoGreen. Chromosome equivalents were determined making use of E. coli MG1655 because the handle and shown in red in comparison with isogenic strains bearing the indicated hda allele shown in blue. Fluorescence intensity (abscissa) is presented in logarithmic scale. (C) Ratio of oriC to TerC on the strain encoding WT or mutant hda. (D) DNA synthesis was measured in reactions containing a supercoiled plasmid bearing oriC in the presence of WT Hda or mutants as described in `Materials and Methods' section. (E) Hydrolysis of ATP bound to DnaA by WT Hda or mutants. (F) DNA synthesis was measured in reactions containing M13Gori1 ssDNA (left) or M13oriC2LB5 supercoiled DNA loop clamp mutant followed by incubation at 30 C for 10 or 20 min as (correct), other reaction components and growing amounts of WT or the loop clamp mutant on the activity of DnaA inside the RIDA assay that measures DNA described in `Materials and Methods' section. (G) Influence of the replication of an oriC-containing plasmid (left) and ATP hydrolysis (suitable).Nucleic Acids Investigation, 2017, Vol. |
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Nucleotides are shown in blue. The F chain of EcHda and also the A chain of SaHda are displayed inside the identical orientation. (B) The superimposed structures of EcHda (chain F, teal) from the Hda?clamp complex and SaHda (3BOS; chain A, orange) collectively with secondary structural elements are shown. ADP in EcHda and CDP in SaHda are shown in magenta and blue sticks, respectively. Each N-terminus of EcHda or SaHda is marked in the bottom with red or blue dots, respectively. (C) Close-up view from the nucleotide-binding web site of the superimposed structures shows the interactions between nucleotide and hydrophobic residues with the lid domain. (D) The superimposed ADP and CDP of EcHda and SaHda, as well as the -phosphate and -phosphate marked by yellow or cyan dots are shown. The sticks are colored as in (B).sensitivity reflects a defect in the interactions among Hda protomers, and/or amongst Hda and DnaA. We also measured the cellular DNA content on the mutant hda strains applying flow cytometry (Figure 6B). The wildtype strain showed big peaks at 2n and 4n positions, as did the hda-Q142A, hda-N144A and hdahook mutants. In contrast, the hda-E126A, hda-E126A-K81R, hda-W156A and hda-Y160A mutants exhibited a single broad peakgreater than 4n (Figure 6B), indicating asynchronous initiation that is definitely consistent with a RIDA defect. We also measured oriC:TerC ratios in these strains (Figure 6C). Compared with the wild-type handle, the hda diaA strains displayed an expected significantly improved oriC:TerC ratio, indicating an excessive frequency of initiations. Whereas the hda-E126A mutant was also elevated, consistent having a RIDA defect, the other hda mutants https://britishrestaurantawards.org/members/singer20europe/activity/356469/ integrated the hda-3900 Nucleic Acids Analysis, 2017, Vol. 45, No.Figure six. In vivo and In vitro analyses of Hda mutants (A) In vivo test of sensitivity of Escherichia coli mutants to hydroxyurea. Just after serial dilution, the sensitivity of E. coli strains encoding mutant Hdas to the indicated concentrations of HU was measured as described in `Materials and Methods' section. Cultures had been normalized for cell density depending on OD600nm before serial dilutions. (B) Flow cytometry evaluation of strains expressing hda alleles. Cells (50 000) in the indicated strains had been analyzed immediately after staining with PicoGreen. Chromosome equivalents were determined making use of E. coli MG1655 because the handle and shown in red in comparison with isogenic strains bearing the indicated hda allele shown in blue. Fluorescence intensity (abscissa) is presented in logarithmic scale. (C) Ratio of oriC to TerC on the strain encoding WT or mutant hda. (D) DNA synthesis was measured in reactions containing a supercoiled plasmid bearing oriC in the presence of WT Hda or mutants as described in `Materials and Methods' section. (E) Hydrolysis of ATP bound to DnaA by WT Hda or mutants. (F) DNA synthesis was measured in reactions containing M13Gori1 ssDNA (left) or M13oriC2LB5 supercoiled DNA loop clamp mutant followed by incubation at 30 C for 10 or 20 min as (correct), other reaction components and growing amounts of WT or the loop clamp mutant on the activity of DnaA inside the RIDA assay that measures DNA described in `Materials and Methods' section. (G) Influence of the replication of an oriC-containing plasmid (left) and ATP hydrolysis (suitable).Nucleic Acids Investigation, 2017, Vol.