Dean A. Jackson

Visualization of replication factories attached to a nucleoskeleton

HeLa cells in early S phase were encapsulated in agarose microbeads, permeabilized, and incubated with biotin-11-dUTP in a physiological buffer. Sites of DNA synthesis were then immunolabeled. As others have found, approximately 150 focal sites of synthesis were visible in each nucleus by light microscopy; they also contained DNA polymerase alpha and proliferating cell nuclear antigen. Electron microscopy of thick resinless sections from which approximately 90% of the chromatin had been removed revealed a similar number of dense, morphologically discrete ovoid bodies strung along a nucleoskeleton. The ovoids remained morphologically and functionally intact despite the removal of most of the chromatin. After 2.5 min of incubation with biotin-11-dUTP, the incorporated analog was associated only with ovoids; after 5 min it began to spread into the adjacent chromatin, which became extensively labeled after 1 hr. This provides visual evidence for polymerization factories fixed to a skeleton, with replication occurring as the template moves through them.

Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III

Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved ‘physiological’ buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br‐UTP, before the resulting Br‐RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that ∼10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only ∼2000 sites (diameter ∼40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or ‘factories’, suggesting that different regions of the nucleus specialize in the transcription of different types of gene.

The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells

The control of RNA synthesis from protein‐coding genes is fundamental in determining the various cell types of higher eukaryotes. The activation of these genes is driven by promoter complexes, and RNA synthesis is performed by an enzyme mega‐complex—the RNA polymerase II ho‐loenzyme. These two complexes are the fundamental components required to initiate gene expression and generate the primary transcripts that, after processing, yield mRNAs that pass to the cytoplasm where protein synthesis occurs. But although this gene expression pathway has been studied intensively, aspects of RNA metabolism remain difficult to comprehend. In particular, it is unclear why >95% of RNA polymerized by polymerase II remains in the nucleus, where it is recycled. To explain this apparent paradox, this review presents a detailed description of nuclear RNA (nRNA) metabolism in mammalian cells. We evaluate the number of active transcription units, discuss the distribution of polymerases on active genes, and assess the efficiency with which the products mature and pass to the cytoplasm. Differences between the behavior of mRNAs on this productive pathway and primary transcripts that never leave the nucleus lead us to propose that these represent distinct populations. We discuss possible roles for nonproductive RNAs and present a model to describe the metabolism of these RNAs in the nuclei of mammalian cells.—Jackson, D. A., Pombo, A., Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells. FASEB J. 14, 242–254 (2000)

Asymmetry of DNA replication fork progression in Werner"s syndrome.

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werners syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.

The structural basis of nuclear function.

Most models for transcription and replication involve polymerases that track along the template. We review here experiments that suggest an alternative in which polymerization occurs as the template slides past a polymerase fixed to a large structure in the eukaryotic nucleus--a factory attached to a nucleoskeleton. This means that higher-order structure dictates how and when DNA is replicated or transcribed.

Chromatin domains and nuclear compartments: establishing sites of gene expression in eukaryotic nuclei

Establishing sites of transcription in the nuclei of higher eukaryotic cells is a very complex process. Before transcription can begin, a series of transcription factors must associate with their recognition motifs, within promoters and more remote activating sequences. Once bound, these factors and associated proteins are believed to form a complex that positions the RNA polymerase holoenzyme so that transcription can commence. As a consequence, active genes assume a specialized chromatin state across regions that define functional domains. Global nuclear architecture appears to stabilize these active domains by providing local environments dedicated to gene expression. As the spatial organization of these sites is unaffected by the removal of most chromatin they must be associated with a structural network. This nucleoskeleton, the associated transcription ‘factories’ and chromatin loops that arise as DNA binds proteins within factories now appear to be fundamental features of nuclear structure in higher eukaryotes. I argue that concentrating proteins needed to perform different steps of RNA synthesis within specialized nuclear compartments will be important in orchestrating events required for efficient gene expression.

Replication and transcription depend on attachment of DNA to the nuclear cage.

SUMMARY When living cells are lysed in a non-ionic detergent and 2m-NaCl, structures are released that resemble nuclei. They contain naked nuclear DNA packaged within a flexible cage of RNA and protein. Since the DNA is supercoiled, it must be intact and looped by attachment to the cage. It is argued that this cage is the active site of the key nuclear functions, transcription and replication: outlying sequences are activated by attachment to polymerases at the cage. This thesis is supported by the close and specific association of nascent RNA with cages, the attachment of active viral sequences (in transformed and productively infected cells) and the attachment of nascent DNA during both normal and repair synthesis.

The attachments of chromatin loops to the nucleoskeleton.

It is widely assumed by cell biologists that chromatin is looped by attachment to some nuclear skeleton. Structural attachments might be mediated through specific sequences; these would be attached in most cells in an organism, underlying the basic structure of the mitotic chromosome and persisting throughout interphase. Functional attachments might also exist, perhaps if active polymerases are attached to the skeleton and replication and transcription occur as DNA is reeled through them. Cells of different tissues--and even cells of the same tissue--would have different attachments of this type. Problems associated with demonstrating these two kinds of attachment are discussed. We find little good evidence for structural attachments and explore the idea that functional attachments are the only kind that exist: functional attachments involving active transcription units might be stable enough to organize chromatin during both interphase and mitosis, but dynamic enough to allow duplication of attached sequences without disrupting loops.

Characterisation of the interaction between WRN, the helicase/exonuclease defective in progeroid Werner's syndrome, and an essential replication factor, PCNA.

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werners syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.

Organization beyond the gene

Abstract Within a nucleus the eukaryotic genome is probably arranged in a complex but highly-ordered fashion. How this organization relates to nuclear function is, at present, a topic of interest.