Uninterrupted MCM2-7 function required for DNA replication fork progression

Little is known about the DNA helicases required for the elongation phase of eukaryotic chromosome replication. Minichromosome maintenance (MCM) protein complexes have DNA helicase activity but have only been functionally implicated in initiating DNA replication. Using an improved method for constructing conditional degron mutants, we show that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae. Like the Escherichia coli dnaB and SV40 T antigen helicases, therefore, the MCM complex is loaded at origins before initiation and is essential for elongation. Restricting MCM loading to the G(1) phase ensures that initiation and elongation occur just once per cell cycle.     

Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase

The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication. A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells. RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity. The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication. These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.     

Inhibition of eukaryotic DNA replication by geminin binding to Cdt1

In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.     

Making sense of eukaryotic DNA replication origins

DNA replication is the process by which cells make one complete copy of their genetic information before cell division. In bacteria, readily identifiable DNA sequences constitute the start sites or origins of DNA replication. In eukaryotes, replication origins have been difficult to identify. In some systems, any DNA sequence can promote replication, but other systems require specific DNA sequences. Despite these disparities, the proteins that regulate replication are highly conserved from yeast to humans. The resolution may lie in a current model for once-per-cell-cycle regulation of eukaryotic replication that does not require defined origin sequences. This model implies that the specification of precise origins is a response to selective pressures that transcend those of once-per-cell-cycle replication, such as the coordination of replication with other chromosomal functions. Viewed in this context, the locations of origins may be an integral part of the functional organization of eukaryotic chromosomes.     

DNA replication-independent silencing in S. cerevisiae

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication. To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase. Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency. Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence.     

Postreplicative formation of cohesion is required for repair and induced by a single DNA break

Sister-chromatid cohesion, established during replication by the protein complex cohesin, is essential for both chromosome segregation and double-strand break (DSB) repair. Normally, cohesion formation is strictly limited to the S phase of the cell cycle, but DSBs can trigger cohesion also after DNA replication has been completed. The function of this damage-induced cohesion remains unknown. In this investigation, we show that damage-induced cohesion is essential for repair in postreplicative cells in yeast. Furthermore, it is established genome-wide after induction of a single DSB, and it is controlled by the DNA damage response and cohesin-regulating factors. We thus define a cohesion establishment pathway that is independent of DNA duplication and acts together with cohesion formed during replication in sister chromatid-based DSB repair.     

Segregating sister genomes: the molecular biology of chromosome separation

During cell division, each daughter cell inherits one copy of every chromosome. Accurate transmission of chromosomes requires that the sister DNA molecules created during DNA replication are disentangled and then pulled to opposite poles of the cell before division. Defects in chromosome segregation produce cells that are aneuploid (containing an abnormal number of chromosomes)-a situation that can have dire consequences. Aneuploidy is a leading cause of spontaneous miscarriages in humans and is also a hallmark of many human cancer cells. Recent work with yeast, Xenopus, and other model systems has provided new information about the proteins that control chromosome segregation during cell division and how the activities of these proteins are coordinated with the cell cycle.     

An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle

An in vitro assay was developed to study the positive factors that regulate the onset of DNA replication during the mammalian cell cycle. Extracts prepared from cells at defined positions in the cell cycle were used to examine the replication of SV40 DNA in a cell free system. Extracts prepared from S phase cells were ten times more efficient at initiating replication at the SV40 origin than were extracts from G1 cells, whereas elongation rates were similar in G1 and S reactions. At a discrete point in the cell cycle, just before the cell's entry into S, an activity appeared that was required, in conjunction with SV40 T antigen, for site specific initiation at the SV40 origin. This factor had a role in unwinding DNA at the replication origin.     

Multienzyme systems of DNA replication

Replication is accomplished by multienzyme systems whose operations are usefully considered in respect to three stages of the process: initiation, elongation, anid termination. 1) Initiation entails synthesis of a short RNA fragment that serves as primer for the elongation step of DNA synthesis. This stage, probed by SS phage DNA templates, reveals three distinctive and highly specific systems in E. coli. The Ml3 DNA utilizes RNA polymerase in a manner that may reflect how plasmid elements are replicated in the cell. The ?X174 DNA does not rely on RNA-polymerase, but requires instead five distinctive proteins which may belong to an apparatus for initiating a host chromosome replication cycle at the origin. The G4 DNA, also independent of RNA polymerase, needs simply the dnaG protein for its distinctive initiation and may thus resemble the system that initiates the replication fragments at the nascent growing fork. In each case it is essential that in vitro the DNA-unwinding protein coat the viral DNA and influence its structure. 2) Elongation is achieved in every case by the multisubunit, holoenzyme form of DNA polymerase III. Copolymerase III, which is an enzyme subunit, and adenosine triphosphate are required to form a proper complex with the primer template but appear dispensable for the ensuing chain growth by DNA polymerase (33). 3) Termination requires excision of the RNA priming fragment, filling of gaps and sealing of interruptions to produce a covalently intact phosphodiester backbone. DNA polymerase I has the capacity for excision and gapfilling and DNA ligase is required for sealing. What once appeared to be a simple DNA polymerase-mediated conversion of a single-strand to a duplex circle (34) is now seen as a complex series of events in which diverse multienzyme systems function. Annoyance with the difficulties in resolving and reconstituting these systems is tempered by the conviction that these are the very systems used ,by the cell in replicating its chromosome and extrachromosomal elements. Thus, understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery.     

Yeast DNA polymerase epsilon participates in leading-strand DNA replication

Multiple DNA polymerases participate in replicating the leading and lagging strands of the eukaryotic nuclear genome. Although 50 years have passed since the first DNA polymerase was discovered, the identity of the major polymerase used for leading-strand replication is uncertain. We constructed a derivative of yeast DNA polymerase epsilon that retains high replication activity but has strongly reduced replication fidelity, particularly for thymine-deoxythymidine 5'-monophosphate (T-dTMP) but not adenine-deoxyadenosine 5'-monophosphate (A-dAMP) mismatches. Yeast strains with this DNA polymerase epsilon allele have elevated rates of T to A substitution mutations. The position and rate of these substitutions depend on the orientation of the mutational reporter and its location relative to origins of DNA replication and reveal a pattern indicating that DNA polymerase epsilon participates in leading-strand DNA replication.     

Bacterial chromosome dynamics

Bacterial chromosomes are highly compacted structures and share many properties with their eukaryote counterparts, despite not being organized into chromatin or being contained within a cell nucleus. Proteins conserved across all branches of life act in chromosome organization, and common mechanisms maintain genome integrity and ensure faithful replication. The principles that underlie chromosome segregation in bacteria and eukaryotes share similarities, although bacteria segregate DNA as it replicates and lack a eukaryote-like mitotic apparatus for segregating chromosomes. This may be because the distances that newly replicated bacterial chromosomes move apart before cell division are small as compared to those in eukaryotes. Bacteria specify positional information, which determines where cell division will occur and which places the replication machinery and chromosomal loci at defined locations that change during cell cycle progression.     

植物增殖细胞核抗原与DNA复制关系的研究现状

为了更深入的对植物增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)进行研究,本研究归纳了PCNA的研究现状,总结了PCNA的结构特征和包括PCNA参与DNA复制、DNA损伤修复和细胞周期调控等在内的PCNA的功能特点。目前有关PCNA的研究以医学和动物体为主,有关植物PCNA研究的报道很少且相对落后,但已有文献指出,PCNA调控DNA复制,参与DNA复制以确保植物体正常生长发育,因此植物PCNA的功能值得继续研究。     

A genome-wide screen identifies genes required for centromeric cohesion

During meiosis, two chromosome segregation phases follow a single round of DNA replication. We identified factors required to establish this specialized cell cycle by examining meiotic chromosome segregation in a collection of yeast strains lacking all nonessential genes. This analysis revealed Sgo1, Chl4, and Iml3 to be important for retaining centromeric cohesin until the onset of anaphase II. Consistent with this role, Sgo1 localizes to centromeric regions but dissociates at the onset of anaphase II. The screen described here provides a comprehensive analysis of the genes required for the meiotic cell cycle and identifies three factors important for the stepwise loss of sister chromatid cohesion.     

Fidelity of mammalian DNA polymerases

The fidelity of copying natural DNA in vitro with each of the three classes of eukaryotic DNA polymerases has been determined. DNA polymerases-beta and -gamma are highly inaccurate, catalyzing noncomplementary single-base substitution at a frequency between 1/3000 and 1/8000. DNA polymerase-alpha is substantially more accurate, with an error rate of 1/30,000. When the error rates of these DNA polymerases are considered in the context of the accuracy of DNA replicative processes in vivo, it seems likely that other factors must exist in mammalian cells which are involved in the accurate replication and maintenance of the genetic information.     

DNA synthesis generates terminal duplications that seal end-to-end chromosome fusions

End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. For more than 70 years, these duplications have been attributed solely to breakage-fusion-bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from Caenorhabditis elegans telomere replication mutants. Genome-level rearrangements revealed fused chromosome ends having interrupted terminal duplications accompanied by template-switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, noncontiguous segments of the genome occurs. Thus, a DNA synthesis-based process may create duplications that seal end-to-end fusions, in the absence of breakage-fusion-bridge cycles.     

Rapid destruction of human Cdc25A in response to DNA damage

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.     

Mechanism of RAD51-dependent DNA interstrand cross-link repair

DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.     

Microbiology. Arresting features of bacterial toxins

Bacteria produce an arsenal of sophisticated toxins that disrupt the normal processes of the host cell, usually by modifying or inactivating host cell proteins. Now, as Coburn and Leong discuss in their Perspective, members of the cytolethal distending toxin (CDT) family have been identified as enzymes that attack DNA (and not protein) within the host cell (Lara-Tejero and Galán). By attacking DNA, perhaps during chromosomal replication, CDTs cause the host cell to halt in G2 phase of the cell cycle.