▪ Abstract The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.

DNA replication in eukaryotic cells

▪ Abstract Genomes are organized into active regions known as euchromatin and inactive regions known as heterochromatin, or silenced chromatin. This review describes contemporary knowledge and models for how silenced chromatin in Saccharomyces cerevisiae forms, functions, and is inherited. In S. cerevisiae, Sir proteins are the key structural components of silenced chromatin. Sir proteins interact first with silencers, which dictate which regions are silenced, and then with histone tails in nucleosomes as the Sir proteins spread from silencers along chromosomes. Importantly, the spreading of silenced chromatin requires the histone deacetylase activity of Sir2p. This requirement leads to a general model for the spreading and inheritance of silenced chromatin or other special chromatin states. Such chromatin domains are marked by modifications of the nucleosomes or DNA, and this mark is able to recruit an enzyme that makes further marks. Thus, among different organisms, multiple forms of repressive chromati...

The Establishment, Inheritance, and Function of Silenced Chromatin in Saccharomyces cerevisiae

A global understanding of the molecular mechanisms underpinning cell biology necessitates an understanding not only of an organism's genome but also of the protein complement encoded within this genome (the proteome). In the past, data regarding an organism's proteome have typically been accumulated piecemeal through studies of a single protein or cell pathway. Genomic methodologies have altered this paradigm: a variety of approaches are now in place by which proteins may be directly analyzed on a proteome-wide scale. Chromatography-coupled mass spectrometry (Gygi et al. 1999; Washburn et al. 2001), large-scale two-hybrid screens (Uetz et al. 2000; Ito et al. 2001; Tong et al. 2002), immunoprecipitation/mass spectrometric analysis of protein complexes (Gavin et al. 2002; Ho et al. 2002), and protein microarray technologies (MacBeath and Schreiber 2000; Zhu et al. 2000, 2001) are yielding unprecedented quantities of protein data. Recent genomic techniques combining microarray technologies with either chromatin immunoprecipitation (Ren et al. 2000; Iyer et al. 2001) or targeted DNA methylation (van Steensel et al. 2001) have been used to globally map binding sites of chromosomal proteins in vivo. Initiatives are even underway to automate and industrialize processes by which protein structures may be solved, potentially providing a library of structural data from which homologous proteins may be modeled (Burley 2000; Montelione 2001).

Although these approaches promise a wealth of information, many fundamental proteomic data sets remain uncataloged. Notably, the subcellular distribution of proteins within any single eukaryotic proteome has never been extensively examined, despite the usefulness and importance of these data. Protein localization is assumed to be a strong indicator of gene function. Localization data are also useful as a means of evaluating protein information inferred from genetic data (e.g., supporting or refuting putative protein interactions suggested from two-hybrid analysis; Ito et al. 2001). Furthermore, the subcellular localization of a protein can often reveal its mechanism of action.

To determine the subcellular localization of a protein, its corresponding gene is typically either fused to a reporter or tagged with an epitope. Reporters and epitope tags are fused routinely to either the N or C termini of target genes, a choice that can be critical in obtaining accurate localization data. Organelle-specific targeting signals (e.g., mitochondrial targeting peptides and nuclear localization signals) are often located at the N terminus (Silver 1991); N-terminal reporter fusions may disrupt these sequences, resulting in anomalous protein localizations. In other cases, C-terminal sequences may be important for proper function and regulation, as recently shown from analysis of the yeast γ-tubulin-like protein Tub4p (Vogel et al. 2001). Gene copy number can also have an impact on the accuracy with which a protein is localized; overexpressed protein products may saturate intracellular transport mechanisms, potentially producing an aberrant subcellular protein distribution. In other cases, weakly expressed single-copy genes may not yield sufficient protein to be visualized, particularly by fluorescence microscopy. The effects of copy number and reporter/tag orientation on protein localization, however, have never been studied in a large data set.

To date, few studies have characterized protein localization on a large scale, primarily because few high-throughput methods exist by which reporter fusions or epitope-tagged proteins can be generated and subsequently localized. Typically, systematic approaches have been used to construct a limited number of chimeric reporter fusions applicable to pilot localization studies. For example, >100 human cDNAs have been cloned as N- and C-terminal gene fusions to spectral variants of green fluorescent protein (GFP) as a means of examining the subcellular localization of these proteins in living cells (Simpson et al. 2000). Thus far, the majority of localization studies have been undertaken in yeast, owing primarily to the fidelity of homologous recombination in Saccharomyces cerevisiae and the concomitant ease with which integrated reporter gene fusions can be generated. As part of a pilot study in S. cerevisiae, Niedenthal et al. (1996) constructed GFP reporter fusions to three unknown open reading frames (ORFs) from yeast Chromosome XIV and subsequently localized these chimeric GFP-fusion proteins by fluorescence microscopy.

In addition to directed cloning methods, strains suitable for localization analysis may be generated through random approaches. Recently, a plasmid-based GFP-fusion library of Schizosaccharomyces pombe DNA was constructed by fusing random fragments of genomic DNA upstream of GFP-coding sequence. Fission yeast cells transformed with this library were subsequently screened for GFP fluorescence, and 250 independent gene products were localized (Ding et al. 2000). In S. cerevisiae, transposon-based methods have been used to generate random lacZ gene fusions (Burns et al. 1994) and epitope-tagged alleles (Ross-MacDonald et al. 1999) for subsequent immunolocalization. Although these transposon-based studies have resulted in the localization of ∼300 yeast proteins, the majority of the S. cerevisiae proteome has remained uncharacterized in regards to its subcellular distribution.

To address this deficiency, we have undertaken the largest analysis to date of protein localization in yeast. Employing high-throughput methods of epitope-tagging and immunofluorescence analysis, our study defines the subcellular localization of 2744 proteins. By integrating these localization data with those previously published, we identify the subcellular localization of >3300 yeast proteins, 55% of the proteome. Building on these data, we have applied a Bayesian system to estimate the intracellular distribution of all 6100 yeast proteins and have further characterized a subset of nuclear proteins both by immunolocalization on surface spread chromosomal preparations and by phenotypic analysis. In total, our findings provide a wealth of insight into protein function, while formally corroborating an expected link between protein function and localization. Furthermore, this study provides experimentally derived localization data for nearly 1000 proteins of previously unknown function, thereby providing, at minimum, a starting point for informed analysis of this previously uncharacterized segment of the proteome.

/pdf/subcellular-localization-of-the-yeast-proteome-3womqw8z62.pdf

Subcellular localization of the yeast proteome

We have sequenced and annotated the genome of the filamentous ascomycete Ashbya gossypii. With a size of only 9.2 megabases, encoding 4718 protein-coding genes, it is the smallest genome of a free-living eukaryote yet characterized. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae. Analysis of this pattern revealed 300 inversions and translocations that have occurred since divergence of these two species. It also provided compelling evidence that the evolution of S. cerevisiae included a whole genome duplication or fusion of two related species and showed, through inferred ancient gene orders, which of the duplicated genes lost one copy and which retained both copies.

http://science.sciencemag.org/content/sci/early/2004/03/04/science.1095781.full.pdf

The Ashbya gossypii Genome as a Tool for Mapping the Ancient Saccharomyces cerevisiae Genome

The MCM proteins are essential replication initiation factors originally identified as proteins required for minichromosome maintenance in Saccharomyces cerevisiae. The best known among them are a family of six structurally related proteins, MCM2-7, which are evolutionally conserved in all eukaryotes. The MCM2-7 proteins form a hexameric complex. This complex is a key component of the prereplication complex that assembles at replication origins during early G1 phase. New evidence suggests that the MCM2-7 proteins may be involved not only in the initiation but also in the elongation of DNA replication. Orchestration of the functional interactions between the MCM2-7 proteins and other components of the prereplication complex by cell cycle-dependent protein kinases results in initiation of DNA synthesis once every cell cycle.

MCM Proteins in DNA Replication

https://www.cell.com/cell/pdf/0092-8674(95)90096-9.pdf

Jay Mitchell

Papers

The multidomain structure of Orc1 p reveals similarity to regulators of DNA replication and transcriptional silencing