PMID:20102606

Sánchez-Romero, MA, Molina, F and Jiménez-Sánchez, A (2010) Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli.BMC Mol. Biol. 11:11

Abstract

<protect>ABSTRACT: BACKGROUND: There 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. RESULTS: We tagged NrdB protein with 3xFLAG 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. CONCLUSIONS: NrdB 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.

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Online version:10.1186/1471-2199-11-11 |} </protect>

Significance

This paper shows that NrdB, and consequently Ribonucleosidediphosphate reductase (RNR), is not evenly distributed throughout the E. coli bacterial cell but it is located in clusters that can be observed as discrete foci by immunofluorescence microscopy.

NrdB foci have the following properties:

1. All foci were always seen within the nucleoid.

2. NrdB foci are located very close to three replication proteins: DnaX or DNA polymerase III τ subunit, helicase DnaB, and SeqA protein.

3. Number of NrdB foci per cell is the same as the number of foci of the three replisome proteins (tested at three different cell cycle periods).

4. NrdB foci appear only in replicating cells and its number decreases as replications end.

The conclusion is that RNR must be associated with the replication complex or replisome. This association would predict that other proteins from the Mathew’s dNTP-synthesizing complex (4) could also be related to the replication fork. This association would require a higher order structure, the hyperstructure (19, 20), to assemble the many functionalities required for precursors biosynthesis, chromosome replication and segregation together with the required membrane structure.

Replication hyperstructure may have unique properties. One of them could be the formation of a discrete pouch that will allow compartmentation of replication precursors by reduction of their diffusion which will permit the precise allosteric regulation of RNR activity, and to attain the high concentrations of the dNTP required for a replicating fork demanding about 1,500 nucleotides per second. Moreover, the tight association among the several functionalities required for precursor biosynthesis, chromosome replication and segregation, and most likely cell division (18), replication hiperstructure would generate the required interconnections that will allow the tight control mechanisms that regulate bacterial cell cycle.

(reference numbers are as in the paper)

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