The metabolic control of DNA replication: mechanism and function.

Metabolism and DNA replication are the two most fundamental biological functions in life. The catabolic branch of metabolism breaks down nutrients to produce energy and precursors used by the anabolic branch of metabolism to synthesize macromolecules. DNA replication consumes energy and precursors for faithfully copying genomes, propagating the genetic material from generation to generation.

The Replicative DnaE Polymerase of Recruits the Glycolytic Pyruvate Kinase (PykA) When Bound to Primed DNA Templates.

The glycolytic enzyme PykA has been reported to drive the metabolic control of replication through a mechanism involving PykA moonlighting functions on the essential DnaE polymerase, the DnaC helicase and regulatory determinants of PykA catalytic activity in . The mutants of this control suffer from critical replication and cell cycle defects, showing that the metabolic control of replication plays important functions in the overall rate of replication. Using biochemical approaches, we demonstrate here that PykA interacts with DnaE for modulating its activity when the replication enzyme is bound to a primed DNA template.

Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication.

Background: In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms.

Multiple links connect central carbon metabolism to DNA replication initiation and elongation in Bacillus subtilis.

DNA replication is coupled to growth by an unknown mechanism. Here, we investigated this coupling by analyzing growth and replication in 15 mutants of central carbon metabolism (CCM) cultivated in three rich media. In about one-fourth of the condition tested, defects in replication resulting from changes in initiation or elongation were detected.

Division-Based, Growth Rate Diversity in Bacteria.

To investigate the nature and origins of growth rate diversity in bacteria, we grew and in liquid minimal media and, after different periods of N-labeling, analyzed and imaged isotope distributions in individual cells with . We find a striking inter- and intra-cellular diversity, even in steady state growth. This is consistent with the strand-dependent, hyperstructure-based hypothesis that a major function of the cell cycle is to generate coherent, growth rate diversity via the semi-conservative pattern of inheritance of strands of DNA and associated macromolecular assemblies.

Interactions of the DnaE polymerase with replisomal proteins modulate its activity and fidelity.

During replication two replicative polymerases function at the replisome to collectively carry out genome replication. In a reconstituted replication assay, PolC is the main polymerase while the lagging strand DnaE polymerase briefly extends RNA primers synthesized by the primase DnaG prior to handing-off DNA synthesis to PolC. Here, we show that (i) the polymerase activity of DnaE is essential for both the initiation and elongation stages of DNA replication, (ii) its error rate varies inversely with PolC concentration, and (iii) its misincorporations are corrected by the mismatch repair system post-replication.

Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis.

Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaEBs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaEBs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex.

Mutations in central carbon metabolism genes suppress defects in nucleoid position and cell division of replication mutants in Escherichia coli.

A genetic link of the carbon metabolism and DNA replication was recently reported for the representative of Gram-negative bacteria, Escherichia coli. Our studies showed that the viability of thermosensitive replication mutants at high temperature can be improved or fully recovered by deleting certain genes of central carbon metabolism (CCM). In order to improve our understanding of this phenomenon, in this study we analyzed the length and nucleoid distribution of suppressed thermosensitive replication mutants.

Combed single DNA molecules imaged by secondary ion mass spectrometry.

Studies of replication, recombination, and rearrangements at the level of individual molecules of DNA are often limited by problems of resolution or of perturbations caused by the modifications that are needed for imaging. The Combing-Imaging by Secondary Ion Mass Spectrometry (SIMS) (CIS) method helps solve these problems by combining DNA combing, cesium flooding, and quantitative imaging via the NanoSIMS 50. We show here that CIS can reveal, on the 50 nm scale, individual DNA fibers labeled with different, nonradioactive isotopes and, moreover, that it can quantify these isotopes so as to detect and measure the length of one or more short nucleic acid fragments associated with a longer fiber.

Genetic response to metabolic fluctuations: correlation between central carbon metabolism and DNA replication in Escherichia coli.

Background: Until now, the direct link between central carbon metabolism and DNA replication has been demonstrated only in Bacillus. subtilis. Therefore, we asked if this is a specific phenomenon, characteristic for this bacterium and perhaps for its close relatives, or a more general biological rule.

Toward a hyperstructure taxonomy.

Bacterial cells contain many large, spatially extended assemblies of ions, molecules, and macromolecules, called hyperstructures, that are implicated in functions that range from DNA replication and cell division to chemotaxis and secretion. Interactions between these hyperstructures would create a level of organization intermediate between macromolecules and the cell itself. To explore this level, a taxonomy is needed.

Genetic evidence for a link between glycolysis and DNA replication.

Background: A challenging goal in biology is to understand how the principal cellular functions are integrated so that cells achieve viability and optimal fitness in a wide range of nutritional conditions.

Methodology/principal Findings: We report here a tight link between glycolysis and DNA synthesis. The link, discovered during an analysis of suppressors of thermosensitive replication mutants in bacterium Bacillus subtilis, is very strong as some metabolic alterations fully restore viability to replication mutants in which a lethal arrest of DNA synthesis otherwise occurs at a high, restrictive, temperature.

Functional taxonomy of bacterial hyperstructures.

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures.

The replicative polymerases PolC and DnaE are required for theta replication of the Bacillus subtilis plasmid pBS72.

Plasmids are the tools of choice for studying bacterial functions involved in DNA maintenance. Here a genetic study on the replication of a novel, low-copy-number, Bacillus subtilis plasmid, pBS72, is reported. The results show that two plasmid elements, the initiator protein RepA and an iteron-containing origin, and at least nine host-encoded replication proteins, the primosomal proteins DnaB, DnaC, DnaD, DnaG and DnaI, the DNA polymerases DnaE and PolC, and the polymerase cofactors DnaN and DnaX, are required for pBS72 replication.

Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis.

In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis.