Role of DNA replication and repair in thymineless death in Escherichia coli

Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.     

Replisome-controlled initiation of DNA replication

A model is presented for initiation of DNA replication according to which a membrane-bound structure, the “replisome”, is brought to maturity on a definite region of the bacterial chromosome. Maturation of replisomes is assumed to be a recurrent process the start and the end of which is marked by initiation of DNA replication. The predictions of the model concerning a temporary inhibition of DNA synthesis are consistent with known but as yet unexplained experimental observations. It is hypothesized that a replisome consists of a protein-RNA complex.     

Escherichia coli RecQ helicase: a player in thymineless death

DNA helicases of the RecQ family are distributed among most organisms and are thought to play important roles in various aspects of DNA metabolism. The founding member of the family, RecQ of Escherichia coli, was identified in a study aimed at clarifying the mechanism of thymineless death, a phenomenon underlying the mechanism for the cytotoxicity of the anticancer drug 5-fluorouracil. The present article is concerned solely with E. coli RecQ and tries to offer an integrated picture of the past and present of its study. Finally a brief discussion is given on how RecQ is involved in thymineless death.     

The initiation of chromosomal DNA replication in eukaryotes

Eukaryotic DNA replication initiates at many sites on each chromosome during the S phase of the cell cycle. Each origin of replication lies in a unique chromosomal environment and can be regulated in different cell types both at the level of utilization and the time of initiation during S phase. In this review, we examine the control and the mechanism of eukaryotic origin function.     

A turnstile for initiation of DNA replication

The progress of a cell through its growth cycle is a multifaceted process; so far we have seen only a glimpse of the complex interplay between the macromolecules performing and regulating the different steps involved. In most organisms, control mechanisms ensure that all chromosomal DNA sequences are replicated once, and only once, between two cell divisions. This enables each division to produce two daughter cells with a genetic content identical to that of their mother. Although the biochemical synthetic processes involved in replicating DNA have been described in detail, our knowledge of the regulatory mechanisms of DNA replication remains scant. In recent experiments with Escherichia coli, new light has been shed on these elusive control mechanisms, and evidence has emerged that may signal an end to our ignorance about this important biological problem.     

Emerging mechanisms of eukaryotic DNA replication initiation

Recent research has focused on proteins important for early steps in replication in eukaryotes, and particularly on Cdc6/Cdc18, the MCMs, and Cdc45. Although it is still unclear exactly what role these proteins play, it is possible that they are analogous to initiation proteins in prokaryotes. One specific model is that MCMs form a hexameric helicase at replication forks, and Cdc6/Cdc18 acts as a ‘clamp-loader’ required to lock the MCMs around DNA. The MCMs appear to be the target of Cdc7-Dbf4 kinase acting at individual replication origins. Finally, Cdc45 interacts with MCMs and may shed light on how cyclin-dependent kinases activate DNA replication.     

Kinetics of thymineless death in Escherichia coli 15 as measured by single-cell observations.

Kinetics of thymineless death for Escherichia coli 15 TAU-bar from plating on solid medium were compared with those from direct observations of single cells under a microscope. The latter method did not involve any physical change of the medium. The kinetics obtained for the two methods were identical. This rules out the assumption that in E. coli 15 TAU-bar death from the thymine deprivation is directly associated with the plating procedure.     

Plasticity of DNA replication initiation in Epstein-Barr virus episomes

In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

Quality control in the initiation of eukaryotic DNA replication

Origins of DNA replication must be regulated to ensure that the entire genome is replicated precisely once in each cell cycle. In human cells, this requires that tens of thousands of replication origins are activated exactly once per cell cycle. Failure to do so can lead to cell death or genome rearrangements such as those associated with cancer. Systems ensuring efficient initiation of replication, while also providing a robust block to re-initiation, play a crucial role in genome stability. In this review, I will discuss some of the strategies used by cells to ensure once per cell cycle replication and provide a quantitative framework to evaluate the relative importance and efficiency of individual pathways involved in this regulation.     

Emerging players in the initiation of eukaryotic DNA replication

Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression.