Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation‐mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N‐terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1‐PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1‐PP1 protects the origin‐binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1‐depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1‐targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.     

The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition

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Dispersion of the cumulus oophorus and dissolution of the zona pellucida in oocytes of DDK and other strains of mice

The time needed in vitro for granulosa cell dispersal with hyaluronidase and for lysis of the zona pellucida with α-chymotrypsin was estimated for unfertilized eggs from three inbred strains of mice, DDK, C57BL, and C3H, and one outbred strain, Q. Granulosa cells were dispersed most rapidly in the DDK and most slowly in the C3H strain. The time needed for dissolution of the zona pellucida was also shortest for DDK eggs and longest for C3H eggs. The results suggest that the high sensitivity to enzymes of the granulosa layer and the zona pellucida of DDK eggs may allow more than one spermatozoon to penetrate through these barriers before the block against polyspermy is completed. The percentage of unfertilized eggs containing spermatozoa under the zona pellucida on the day of copulation was abnormally high in matings of females from the DDK inbred strain with males from DDK and C57BL strains. This may suggest some disturbances in establishing contact between the egg plasma membrane and fertilizing spermatozoa.     

The osteoprotective role of USP26 in coordinating bone formation and resorption

Bone homeostasis is maintained through a balance of bone formation by osteoblasts and bone resorption by osteoclasts. Ubiquitin-specific proteases (USPs) are involved in regulating bone metabolism by preserving bone formation or antagonizing bone resorption. However, the specific USPs that maintain bone homeostasis by orchestrating bone formation and bone resorption simultaneously are poorly understood. Here, we identified USP26 as a previously unknown regulator of bone homeostasis that coordinates bone formation and resorption. Mechanistically, USP26 stabilizes β-catenin to promote the osteogenic activity of mesenchymal cells (MSCs) and impairs the osteoclastic differentiation of bone myelomonocytes (BMMs) by stabilizing inhibitors of NF-κBα (IκBα). Gain-of-function experiments revealed that Usp26 supplementation significantly increased bone regeneration in bone defects in aged mice and decreased bone loss resulting from ovariectomy. Taken together, these data show the osteoprotective effect of USP26 via the coordination of bone formation and resorption, suggesting that USP26 represents a potential therapeutic target for osteoporosis.Subject terms: Deubiquitylating enzymes, Deubiquitylating enzymes, Endocrine system and metabolic diseases, Immunopathogenesis     

The role of electrostatic interactions in calmodulin-peptide complex formation

The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from approximately -5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.     

The survivin/Aurora B complex: its role in coordinating tension and attachment

Proper chromosome segregation relies on the action of the spindle checkpoint. Recent data have shown that the chromosomal passenger proteins survivin and Aurora B play an important auxiliary role in spindle checkpoint surveillance. Knock-down experiments in human cells indicate that the function of the survivin/Aurora B complex is required to correct improper microtubule-kinetochore interactions. Combined data of four different groups show that the survivin/Aurora B complex is not an integral component of the spindle checkpoint, but it enables the cell to communicate lack of tension back to the attached microtubules. Moreover, they show that the affinity of BubR1 for kinetochores is directly influenced by the absence or presence of the survivin/Aurora B complex. These functions of the survivin/Aurora B complex are essential for chromosome biorientation, a prerequisite for proper chromosome segregation. As such, this complex plays an important role in the maintenance of a stable genome.     

The role of miRNAs in complex formation and control

microRibonucleic acid (miRNAs) are small regulatory molecules that act by mRNA degradation or via translational repression. Although many miRNAs are ubiquitously expressed, a small subset have differential expression patterns that may give rise to tissue-specific complexes. MOTIVATION: This work studies gene targeting patterns amongst miRNAs with differential expression profiles, and links this to control and regulation of protein complexes. RESULTS: We find that, when a pair of miRNAs are not expressed in the same tissues, there is a higher tendency for them to target the direct partners of the same hub proteins. At the same time, they also avoid targeting the same set of hub-spokes. Moreover, the complexes corresponding to these hub-spokes tend to be specific and nonoverlapping. This suggests that the effect of miRNAs on the formation of complexes is specific.     

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.     

The role of hydrophobic interactions in ankyrin-spectrin complex formation

Spectrin and ankyrin are the key components of the erythrocyte cytoskeleton. The recently published crystal structure of the spectrin-ankyrin complex has indicated that their binding involves complementary charge interactions as well as hydrophobic interactions. However, only the former is supported by biochemical evidence. We now show that nonpolar interactions are important for high affinity complex formation, excluding the possibility that the binding is exclusively mediated by association of distinctly charged surfaces. Along these lines we report that substitution of a single hydrophobic residue, F917S in ankyrin, disrupts the structure of the binding site and leads to complete loss of spectrin affinity. Finally, we present data showing that minimal ankyrin binding site in spectrin is formed by helix 14C together with the loop between helices 15 B/C.     

Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation

The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum . Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae o rigin r ecognition c omplex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.     

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G₁ transition and for pre-RC maintenance in G₁ phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.     

The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis

Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.     

Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one‐cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one‐cell embryo can be an important model for understanding the regulation of DNA synthesis. J. Cell. Biochem. 107: 214–223, 2009. © 2009 Wiley‐Liss, Inc.     

The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation

Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers. This process is dependent on Cdc6, which is recruited to unlicensed origins. Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers. This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed. Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA. We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition. These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication.     

The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis

Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.     

The role of SMARCAL1 in replication fork stability and telomere maintenance

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.