Ribonucleoside diphosphate reductase (RNR) of
The best-known defective RNR mutant in
The RNR101 protein is inactivated at 42°C
These results are consistent with RNR having a thermoresistance period due to protection by some subcellular structure. This enzyme has been proposed to be part of a complex for the biosynthesis of dNTP (Mathews, 1993) therefore the association with this complex might explain such protection. We have proposed that, as a component of the replication hyperstructure, the RNR101 protein would be protected from thermal inactivation and that this would suffice to allow chromosome replication for 50 min in restrictive conditions (Guzmán et al., 2002; 2003, Molina & Skarstad, 2004; Guarino et al., 2007a; 2007b; Riola et al., 2007).
Supporting this model, RNR has been colocalized with the replisome-associated proteins DnaB helicase and DNA polymerase τ subunit, and with the fork-associated protein SeqA (Fig. 2) (Sánchez-Romero et al., 2010).
Furthermore, a hyperstructure containing RNR101 impairs replication fork progression even at the permissive temperature (Guarino et al., 2007a). Arrest of replication forks is known to cause double-strand breaks, DSBs (Bierne & Michel, 1994; Kuzminov, 1995). We have shown that the number of DSBs in the
|Relevant phenotype||% linear DNA a|
It is intriguing that rifampicin or cloramphenicol addition, as well as the presence of a
In studying replication in the
Consequently, we propose that a reduction in the number of forks replicating the chromosome results in an improvement in the quality of replication that allows the deficient replication hyperstructure of the
2. Reduction in the overlap of replication rounds improves fork progression at the restrictive temperature in a
We have previously shown that, due to an elongation of the replication period lasting more than twice the cell cycle at 30°C (
2.1. Experimental approach
In contrast with eukaryotic organisms, the time required to replicate a single chromosome (
As explained above, the overlap of the replication rounds depends on two parameters, the generation time, τ, and the
2.2. Increasing the generation time
As expected from growing the bacteria in a carbon source different from glucose, a lengthening of the generation time and a lowering in the number of overlapped replication rounds,
2.3. Reducing the C period
2.3.1. By the presence of
dnaA defective alleles
The presence of
When incubated for 4 h at 42°C in the presence of cephalexin, the DNA content per cell in
We have verified that the number of overlapped replication forks per chromosome at 30°C could be also lowered by introducing
These results suggest that a lowering in the number of replication forks running along the chromosome could improve the progression of replication in the
In addition, over-expression of the
It has been shown that the
2.3.2. By increasing the number of copies of the
2.3.3. By deleting the DARS sequence
The DnaA protein is a member of the AAA+ ATPase family and has an exceptionally high affinity for ATP/ADP (Sekimuzu et al., 1987; Kaguni, 2006). The level of cellular ATP-DnaA oscillates during the replication cycle, peaking around the time of initiation (Kurokawa et al., 1999).
Katayama's group has recently found two chromosomal intergenic regions termed DARS1 and DARS2 (
Our data show that decreasing the number of replication rounds (
Furthermore, a lower availability of wild type DnaA protein induced by the presence of extra copies of the
These observations, together with our data, are consistent with the idea that the progression of replication forks is not merely responsive to elongation factors (dNTP pools or proteins engaged in elongation) but also to the number of forks running along the chromosome. We suggest that the best explanation for the reduction of the
3. Stalled multifork chromosomes as the cause of aberrant DNA segregation and cell death in the
nrdA101 mutant at the restrictive temperature
Cell viability was studied in all the growth media and strains described above. Cells were grown at 30°C and when the cultures reached mid-logarithmic phase (about 0.1 OD550), an aliquot of each culture was incubated at 42°C and the number of viable cells were estimated by serial dilution and plating on rich medium at 30°C. Viability is expressed relative to the onset of treatment. Growing
Nucleoid segregation analysis was performed in aliquots of the cultures incubated at 42°C in the presence of cephalexin (50 µg/ml) for 4 hours plus, during the last 20 min, chloramphenicol addition(200µg/ml) to condense nucleoids. Micrographs of DAPI stained cells show a high number of cells containing an abnormal number of nucleoids randomly distributed along the filaments (Fig. 5) (Riola et al., 2007). An increased number of cells containing normal and well-segregated nucleoids were found in cells grown in arabinose or in glycerol (Fig. 5). The anomalous number and distribution of nucleoids found in the
The above results reveal a good correlation between the overlap of replication rounds and aberrant nucleoid segregation and cell lethality. This correlation is consistent with the hypothesis that these problems are associated with a highly forked chromosome structure. The detrimental effects of such chromosomes are reduced or eliminated by any environmental or genetic modification that reduces replication overlap. We therefore suggest that the observed morphological alterations of
overlaps, stalled forks have less opportunity to be repaired and restarted and this interferes with subsequent forks. This results in chromosomal abnormalities, disrupted chromosome and nucleoid segregation, loss of cell division, and, finally, cell death.
DNA topology has been found to play an important role in the segregation of duplicated chromosomes (Dasgupta et al., 2000; Holmes & Cozarelli, 2000). Consequently, a disturbed DNA topology due to a highly forked chromosome structure, could contribute to the altered nucleoid segregation observed in the
4. The number of replication rounds in the chromosome limits the replication rate of individual forks
It is difficult to decide whether the reduction in the number of forks is the consequence of an increased replication rate (as
The second proposition is that the elongation rate increased as a consequence of the reduction of the number of forks or the replication overlap. This reduction in the number of the forks would be caused by the deficiency of any factor required for the initiation step since this would result in the delay of the initiation of replication.
In the above work, we have shown that a decrease in the growth rate of the
An unified explanation for all the results presented here is difficult to find. Clearly though, the underlying mechanism should explain the precise correlation between initiation and elongation that tunes DNA replication to any environmental circumstance. Whatever the nature of this mechanism, reduction in the number of forks per chromosome or decreased overlapping of consecutive replication rounds might increase the elongation rate by providing
a better overall chromosome structure, including discrete regional organization and supercoiling domains,
an increased availability of a limiting constituent required for replication and/or for segregation, and
an increased time for the repair and restart of a stalled fork so as to avoid collision with the next fork.
This homeostatic regulation between the numbers and velocities of forks would also explain how the replication rate compensates for widely varying replication origins and activities in eukaryotes (Conti et al., 2007).
5. Balance between the number of origins and elongation rates as a general regulatory mechanism in the control of eukaryotic cell cycle
In eukaryotic cells, the DNA replication program is organized according to multiple tandem replicons that span each chromosome. Each replicon is replicated bidirectionally by a pair of replication forks that increase their rates up to three fold towards the end of S phase. Furthermore, the rate of the replication fork progression varies up to ten-fold or more depending on the distance between origins in different conditions or cell types (Housman & Huberman, 1975; reviewed in Herrick, 2010). Two replication regimes with distinct kinetics govern duplication of the genome: in the first half of the S phase, when the gene-rich euchromatin is predominantly replicated, the density of the activated replication origins steadily increases to about twice the initial value; during the second half of the S phase, when the gene-poor heterochromatin tends to be replicated, the density of active replication origins increases substantially by about ten fold (Herrick & Bensimon, 2008). It has been proposed that this mechanism would guarantee the rapid and complete duplication of the genome. Nevertheless, in mammalian cells the relationship between origin activation, the size of replicons (50-300kb) and the existence of multiple potential origins remains to be elucidated (Herrick, 2010).
The efficient duplication of the eukaryotic genome depends on the orderly activation of the origins, estimated to be ten thousand, and on the proper progression of their forks. The coordinated activation of origins is insufficient on its own to account for timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Therefore the coordination and compensation between origin spacing and fork progression may be one of the mechanisms for the complete duplication of the genome in the limited amount of time of the S phase. By using a single-molecule approach based on molecular combing, the interorigin distances and replication fork velocities over extensive regions of the genome have been measured in both primary keratinocytes and cancer cells (Conti et al., 2007). This study provides evidence for the direct correlation between the interorigin distances and the replication rates, insofar as the further the origins are from one another, the faster the forks progress. These results are in agreement with the results of this and other studies of
Figure 3 in Conti et al., 2007 shows a significant linear correlation between these two parameters in eukaryotic cells, consistent with a biological mechanism that coordinates replication fork progression with interorigin distance. The mechanism that allows replication forks to adjust their speed is unknown. Nevertheless the possibilities for the nature of this mechanism are similar to the ones proposed above for
6. Concluding remarks
In this work we show that reducing the number of replication forks per chromosome in
variations in the availability of some limiting hyperstructure component which might lead to assembly of an inefficient hyperstructure when a high number of forks compete for this component, or
the structural constraints caused by a chromosome undergoing several rounds of replication running at the same time.
Results from other research groups, reviewed above, and comparison with DNA replication in eukaryotes provide further evidence that, in widely different systems, the initiation and the elongation of chromosome replication are not independent processes.
We are very grateful to Kirsten Skarstad and Tsutomu Katayama for bacterial strains and plasmids. We especially thank Encarna Ferrera for her technical help. This work was supported by grant BFU2007-63942 from the Ministerio de Ciencia e Innovación. IS, CM and MAS-R acknowledge the studentship from Junta de Extremadura.
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