Regulating DNA Replication in Eukarya
TL;DR: Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed.
Abstract: DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication.
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TL;DR: The reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides is described, which defines the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.
Abstract: Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.
439 citations
TL;DR: New insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.
Abstract: DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.
426 citations
TL;DR: The authors' increasingly molecular understanding of the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle and their regulation is reviewed.
Abstract: The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation.
305 citations
TL;DR: The general nature of the DNA replication machinery is outlined, but also points out important and key differences.
Abstract: The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle.
289 citations
Cites background from "Regulating DNA Replication in Eukar..."
...These issues are discussed in Siddiqui et al. (2013) and Zielke et al. (2013)....
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...Cellcycle phases and their regulation are explained in McIntosh and Blow (2012), Siddiqui et al. (2013), Skarstad and Katayama (2013), and Zielke et al. (2013)....
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TL;DR: This work has shown that through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions.
224 citations
Cites background from "Regulating DNA Replication in Eukar..."
...PCNA is an essential co-factor for DNA polymerases during replication (O’Donnell et al., 2013; Siddiqui et al., 2013)....
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...DNA replication is initiated at distinct replication origins, which are marked by binding of the pre-replicative protein complex in G1 phase of the cell cycle (O’Donnell et al., 2013; Siddiqui et al., 2013)....
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...DNA Replication DNA replication is initiated at distinct replication origins, which are marked by binding of the pre-replicative protein complex in G1 phase of the cell cycle (O’Donnell et al., 2013; Siddiqui et al., 2013)....
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References
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TL;DR: A comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle is created, and it is found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins.
Abstract: We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures sync...
5,176 citations
"Regulating DNA Replication in Eukar..." refers background in this paper
...Whereas SBF activates transcription of Cln cyclins (Nasmyth and Dirick 1991; Spellman et al. 1998; Eser et al. 2011), MBF promotes transcription of Clb5 along with other replication genes (Lowndes et al. 1992; Koch et al. 1993; Spellman et al. 1998; Eser et al. 2011)....
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TL;DR: There is now evidence that most cancers may indeed be genetically unstable, but that the instability exists at two distinct levels, and recognition and comparison of these instabilities are leading to new insights into tumour pathogenesis.
Abstract: Whether and how human tumours are genetically unstable has been debated for decades. There is now evidence that most cancers may indeed be genetically unstable, but that the instability exists at two distinct levels. In a small subset of tumours, the instability is observed at the nucleotide level and results in base substitutions or deletions or insertions of a few nucleotides. In most other cancers, the instability is observed at the chromosome level, resulting in losses and gains of whole chromosomes or large portions thereof. Recognition and comparison of these instabilities are leading to new insights into tumour pathogenesis.
4,121 citations
"Regulating DNA Replication in Eukar..." refers background in this paper
...2010), a phenomenon observed in many human cancers (Lengauer et al. 1998)....
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...This leads to gene amplification and promotes genome instability (Green et al. 2010), a phenomenon observed in many human cancers (Lengauer et al. 1998)....
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TL;DR: Genetic evidence suggests that tumour cells may also require specific interphase CDKs for proliferation, and selective CDK inhibition may provide therapeutic benefit against certain human neoplasias.
Abstract: Tumour-associated cell cycle defects are often mediated by alterations in cyclin-dependent kinase (CDK) activity. Misregulated CDKs induce unscheduled proliferation as well as genomic and chromosomal instability. According to current models, mammalian CDKs are essential for driving each cell cycle phase, so therapeutic strategies that block CDK activity are unlikely to selectively target tumour cells. However, recent genetic evidence has revealed that, whereas CDK1 is required for the cell cycle, interphase CDKs are only essential for proliferation of specialized cells. Emerging evidence suggests that tumour cells may also require specific interphase CDKs for proliferation. Thus, selective CDK inhibition may provide therapeutic benefit against certain human neoplasias.
3,146 citations
"Regulating DNA Replication in Eukar..." refers background in this paper
...Several cyclin subunits and CDK2 can be knocked out in mice (Sherr and Roberts 2004; Malumbres and Barbacid 2009), suggesting significant redundancy among the functions of cyclin–CDK complexes....
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TL;DR: This work has witnessed an explosion in understanding of DNA damage sensing, signaling, and the complex interplay between protein phosphorylation and the ubiquitin pathway employed by the DDR network to execute the response to DNA damage.
1,639 citations
TL;DR: A better understanding of the ubiquitylation machinery will provide new insights into the regulatory biology of cell-cycle transitions and the development of anti-cancer drugs.
Abstract: A driving force of the cell cycle is the activation of cyclin-dependent kinases (CDKs), the activities of which are controlled by the ubiquitin-mediated proteolysis of key regulators such as cyclins and CDK inhibitors. Two ubiquitin ligases, the SKP1-CUL1-F-box-protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C), are responsible for the specific ubiquitylation of many of these regulators. Deregulation of the proteolytic system might result in uncontrolled proliferation, genomic instability and cancer. Cumulative clinical evidence shows alterations in the ubiquitylation of cell-cycle regulators in the aetiology of many human malignancies. A better understanding of the ubiquitylation machinery will provide new insights into the regulatory biology of cell-cycle transitions and the development of anti-cancer drugs.
1,365 citations
"Regulating DNA Replication in Eukar..." refers background in this paper
...During prometaphase, Cdc2 and Plk1 cooperate to phosphorylate these inhibitors, presenting them for SCFb-TrCP-mediated degradation, thus relieving the inhibition of the APC/C and promoting mitosis (Nakayama and Nakayama 2006)....
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