Felipe Molina

Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism

Replication from the origin of Escherichia coli has traditionally been visualized as two replisomes moving away from each other, each containing a leading and a lagging strand polymerase. Fluorescence microscopy studies of tagged polymerases or forks have, however, indicated that the polymerases may be confined to a single location (or a few locations in cells with overlapping replication cycles). Here, we have analysed the exact replication patterns of cells growing with four different growth and replication rates, and compared these with the distributions of SeqA foci. The SeqA foci represent replication forks because the SeqA protein binds to the newly formed hemimethylated DNA immediately following the forks. The results show that pairs of forks originating from the same origin stay coupled for most of the cell cycle and thus support the replication factory model. They also suggest that the factories consisting of four polymerases are, at the time immediately after initiation, organized into higher order structures consisting of eight or 12 polymerases. The organization into replication factories was lost when replication forks experienced a limitation in the supply of nucleotides or when the thymidylate synthetase gene was mutated. These results support the idea that the nucleotide synthesis apparatus co‐localizes with the replisomes forming a ‘hyperstructure’ and further suggest that the integrity of the replication factories and hyperstructures is dependent on nucleotide metabolism.

Deletion of the datA site does not affect once-per-cell-cycle timing but induces rifampin-resistant replication

In Escherichia coli, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in deltadatA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.

Genetic Instability of Heterozygous, Hybrid, Natural Wine Yeasts

ABSTRACT We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2R/cyh2R mutants resistant to high levels of cycloheximide can be directly isolated from cyh2S/cyh2S wine yeasts. Heterozygous cyh2R/cyh2S hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2R/cyh2R homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.

Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli

BackgroundThere has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm. Consequently we were interested in the localization of RNR protein and its relationship with other replication proteins.ResultsWe tagged NrdB protein with 3×FLAG epitope and detected its subcellular location by immunofluorescence microscopy. We found that this protein is located in nucleoid-associated clusters, that the number of foci correlates with the number of replication forks at any cell age, and that after the replication process ends the number of cells containing NrdB foci decreases.We show that the number of NrdB foci is very similar to the number of SeqA, DnaB, and DnaX foci, both in the whole culture and in different cell cycle periods. In addition, interfoci distances between NrdB and three replication proteins are similar to the distances between two replication protein foci.ConclusionsNrdB is present in nucleoid-associated clusters during the replication period. These clusters disappear after replication ends. The number of these clusters is closely related to the number of replication forks and the number of three replication protein clusters in any cell cycle period. Therefore we conclude that NrdB protein, and most likely RNR protein, is closely linked to the replication proteins or replisome at the replication fork. These results clearly support the replication hyperstructure model.

CHROMOSOMAL INSERTIONS LOCALIZED AROUND ORIC AFFECT THE CELL CYCLE IN ESCHERICHIA COLI

The present work reports the effects of localized insertions around the origin of Escherichia coli chromosome, oriC, on cell cycle parameters. These insertions cause an increase of the C period with an inverse correlation to the distance from oriC. In addition, Omega insertion near oriC causes an increase in the number of replication forks per chromosome, n, and Tn10 insertion causes a decrease in growth rate. We found that the same insertion positioned in another region of the chromosome, outside of oriC, has a negligible effect on the C period. Marker frequency analysis suggests a slower replication velocity along the whole chromosome. We propose that the insertions positioned at less than 2 kbp from oriC could create a structural alteration in the origin of replication that would result in a longer C period. Flow cytometry reveals that asynchrony is not associated with these alterations.

Organization of ribonucleoside diphosphate reductase during multifork chromosome replication in Escherichia coli

Ribonucleoside diphosphate reductase (RNR) is located in discrete foci in a number that increases with the overlapping of replication cycles in Escherichia coli. Comparison of the numbers of RNR, DnaX and SeqA protein foci with the number of replication forks at different growth rates reveals that fork : focus ratios augment with increasing growth rates, suggesting a higher cohesion of the three protein foci with increasing number of forks per cell. Quantification of NrdB and SeqA proteins per cell showed: (i) a higher amount of RNR per focus at faster growth rates, which sustains the higher cohesion of RNR foci with higher numbers of forks per cell; and (ii) an equivalent amount of RNR per replication fork, independent of the number of the latter.

Determination of cell cycle parameters by flow cytometry in bacteria: practical considerations

The cell cycle of slowly growing bacteria consists of three phases that resemble the eukaryotic cell cycle: the preinitiation, replication and post-replication periods, termed B, C and D, respectively. However, bacteria are also capable of very rapid growth, whereas the replication period remains long and hence the B period disappears and a round of replication is triggered before the previous one is finished. Several frequently used methods yield an incomplete analysis of these rapidly growing bacterial cultures. However, DNA distributions obtained by flow cytometry can be used to fully unravel the bacterial cell cycle independently of the duration of each period. In the present work, we have compared the accuracy of the cell cycle parameters estimation in different cultures. Whereas, for a given DNA per cell content, the accuracy of the C period determination by flow cytometry is essentially independent of the cell cycle, the precision of the D period estimation is inversely correlated with the number of replication forks per chromosome. We have established a data analysis routine to estimate the accuracy of the cell cycle parameters determination.