The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

Structural basis for inhibition of the replication licensing factor Cdt1 by geminin

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. Here we describe the crystal structure of the mouse geminin-Cdt1 complex using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.     

The wee1 protein kinase is required for radiation-induced mitotic delay.

Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions. In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage. Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis. Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements. We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway. Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1.     

A novel nuclear export activity in HIV-1 matrix protein required for viral replication

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.     

MDC1 is required for the intra-S-phase DNA damage checkpoint

MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.     

MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans

The germ line is an immortal cell lineage that is passed indefinitely from one generation to the next. To identify the genes that are required for germline immortality, we isolated Caenorhabditis elegans mutants with mortal germ lines--worms that can reproduce for several healthy generations but eventually become sterile. One of these mortal germline (mrt) mutants, mrt-2, exhibits progressive telomere shortening and accumulates end-to-end chromosome fusions in later generations, indicating that the MRT-2 protein is required for telomere replication. In addition, the germ line of mrt-2 is hypersensitive to X-rays and to transposon activity. Therefore, mrt-2 has defects in responding both to damaged DNA and to normal double-strand breaks present at telomeres. mrt-2 encodes a homologue of a checkpoint gene that is required to sense DNA damage in yeast. These results indicate that telomeres may be identified as a type of DNA damage and then repaired by the telomere-replication enzyme telomerase.     

DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage.

Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.     

The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.     

SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer.

Non-clathrin coated vesicles have been implicated in early steps of intercompartmental transport. A distinct set of coat proteins are peripherally associated with the exterior of purified mammalian intra-Golgi transport vesicles. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (beta-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat. Here we describe the characterization of SEC21, an essential gene required for protein transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat protein plays an essential role in intercompartmental transport.     

ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response

Nijmegen breakage syndrome (NBS) is characterized by extreme radiation sensitivity, chromosomal instability and cancer. The phenotypes are similar to those of ataxia telangiectasia mutated (ATM) disease, where there is a deficiency in a protein kinase that is activated by DNA damage, indicating that the Nbs and Atm proteins may participate in common pathways. Here we report that Nbs is specifically phosphorylated in response to gamma-radiation, ultraviolet light and exposure to hydroxyurea. Phosphorylation of Nbs mediated by gamma-radiation, but not that induced by hydroxyurea or ultraviolet light, was markedly reduced in ATM cells. In vivo, Nbs was phosphorylated on many serine residues, of which S343, S397 and S615 were phosphorylated by Atm in vitro. At least two of these sites were underphosphorylated in ATM cells. Inactivation of these serines by mutation partially abrogated Atm-dependent phosphorylation. Reconstituting NBS cells with a mutant form of Nbs that cannot be phosphorylated at selected, ATM-dependent serine residues led to a specific reduction in clonogenic survival after gamma-radiation. Thus, phosphorylation of Nbs by Atm is critical for certain responses of human cells to DNA damage.     

SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans

Ionotropic glutamate receptors (iGluRs) mediate most excitatory synaptic signalling between neurons. Binding of the neurotransmitter glutamate causes a conformational change in these receptors that gates open a transmembrane pore through which ions can pass. The gating of iGluRs is crucially dependent on a conserved amino acid that was first identified in the 'lurcher' ataxic mouse. Through a screen for modifiers of iGluR function in a transgenic strain of Caenorhabditis elegans expressing a GLR-1 subunit containing the lurcher mutation, we identify suppressor of lurcher (sol-1). This gene encodes a transmembrane protein that is predicted to contain four extracellular beta-barrel-forming domains known as CUB domains. SOL-1 and GLR-1 are colocalized at the cell surface and can be co-immunoprecipitated. By recording from neurons expressing GLR-1, we show that SOL-1 is an accessory protein that is selectively required for glutamate-gated currents. We propose that SOL-1 participates in the gating of non-NMDA (N-methyl-D-aspartate) iGluRs, thereby providing a previously unknown mechanism of regulation for this important class of neurotransmitter receptor.     

A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.     

Differentiated parental DNA strands confer developmental asymmetry on daughter cells in fission yeast

The two strands of the DNA molecule are complementary but not identical. Hence, upon semiconservative replication, different parental DNA strands are segregated to daughter cells. A molecular analysis suggests that the process of fission yeast mating-type interconversion uses asymmetry of the DNA strands to generate a regular lineage of cellular differentiation.     

Requirement for the replication protein SSB in human DNA excision repair

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.     

Role for a Drosophila Myb-containing protein complex in site-specific DNA replication

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.