1887

Abstract

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, cells carrying the allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in the mutant. We found that inactivation of the DnaA protein by using several alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the mutant at 42 °C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of sequence increases the relative amount of DNA synthesis of the mutant at 42 °C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

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2011-07-01
2019-10-22
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Flow cytometry profile of and strains after 4 h at 42 °C with cephalexin in the absence (dashed line) or in the presence (solid line) of rifampicin. [PDF](25 kb) (a) Phase-contrast microscopy of cells from the and strains after different times at 42 °C to study cell division. (b) Fluorescence microscopy of Hoechst-stained cells from the and strains after different times at 42 °C in the presence of cephalexin to study DNA segregation. Cells were fixed as for the flow cytometry. To stain cells with Hoechst, 1 ml of each fixed sample was centrifuged, washed with PBS, and resuspended in 10 μl Hoechst. The sample was then separately spread onto a microscope slide, dried and treated with 10 μl polylysine. Cells were visualized by phase-contrast and fluorescence microscopy. A Nikon Eclipse E600 microscope with a Hamamatsu C4742-95 camera was used to capture the micrographs. [PDF](250 kb) Western blot analysis of the R1 subunit (NrdA) in the and strains after different times at 42 °C. β-Galactosidase levels were used as a loading control. [PDF](150 kb)

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Flow cytometry profile of and strains after 4 h at 42 °C with cephalexin in the absence (dashed line) or in the presence (solid line) of rifampicin. [PDF](25 kb) (a) Phase-contrast microscopy of cells from the and strains after different times at 42 °C to study cell division. (b) Fluorescence microscopy of Hoechst-stained cells from the and strains after different times at 42 °C in the presence of cephalexin to study DNA segregation. Cells were fixed as for the flow cytometry. To stain cells with Hoechst, 1 ml of each fixed sample was centrifuged, washed with PBS, and resuspended in 10 μl Hoechst. The sample was then separately spread onto a microscope slide, dried and treated with 10 μl polylysine. Cells were visualized by phase-contrast and fluorescence microscopy. A Nikon Eclipse E600 microscope with a Hamamatsu C4742-95 camera was used to capture the micrographs. [PDF](250 kb) Western blot analysis of the R1 subunit (NrdA) in the and strains after different times at 42 °C. β-Galactosidase levels were used as a loading control. [PDF](150 kb)

PDF

Flow cytometry profile of and strains after 4 h at 42 °C with cephalexin in the absence (dashed line) or in the presence (solid line) of rifampicin. [PDF](25 kb) (a) Phase-contrast microscopy of cells from the and strains after different times at 42 °C to study cell division. (b) Fluorescence microscopy of Hoechst-stained cells from the and strains after different times at 42 °C in the presence of cephalexin to study DNA segregation. Cells were fixed as for the flow cytometry. To stain cells with Hoechst, 1 ml of each fixed sample was centrifuged, washed with PBS, and resuspended in 10 μl Hoechst. The sample was then separately spread onto a microscope slide, dried and treated with 10 μl polylysine. Cells were visualized by phase-contrast and fluorescence microscopy. A Nikon Eclipse E600 microscope with a Hamamatsu C4742-95 camera was used to capture the micrographs. [PDF](250 kb) Western blot analysis of the R1 subunit (NrdA) in the and strains after different times at 42 °C. β-Galactosidase levels were used as a loading control. [PDF](150 kb)

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