The regulation of pre-replicative complex formation in the budding yeast cell cycle

DNA replication must occur once and only once in every cell cycle to ensure that mitosis produces two daughter cells with the same complement of genomic DNA. Initiation of DNA replication depends upon pre-replicative complex (pre-RC) assembly at origins during Gl. The pre-RC includes a six-subunit origin recognition complex (ORC) and Cdc6p which together load a complex of the Mcm2-7p family of putative helicases onto chromatin. Only one ORC subunit, Orc6p, is not required for binding of the others to origins in vitro. Using a temperature sensitive orc6 mutant we have shown in vivo that although Orc6p is dispensable for binding of other ORC subunits to chromatin, it is essential for pre-RC formation and may function as an "adapter" between ORC and other pre-RC components. The activity of cyclin dependent kinases (CDKs) regulates pre-RC formation in the cell cycle. CDKs inhibit the formation of pre-RCs during S phase, G2 and M phase, suggesting that inactivation of CDKs at the end of mitosis is essential for pre- RC formation in Gl. Other mitotic events may also be important for pre-RC formation. In particular, the anaphase promoting complex (APC/C), which targets mitotic proteins for proteolysis and the mitotic exit network which activates a mitotic phosphatase, Cdcl4p, have been implicated in pre-RC formation. We have found that inactivation of CDKs during mitosis bypasses the requirement for the APC/C and the mitotic exit network in DNA replication. This suggests that the only essential role for these proteins for pre-RC formation is to bring about CDK inactivation at the end of mitosis

Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions

The matrix (M1) protein of influenza A virus is a multifunctional protein that plays essential structural and functional roles in the virus life cycle. It drives virus budding and is the major protein component of the virion, where it forms an intermediate layer between the viral envelope and integral membrane proteins and the genomic ribonucleoproteins (RNPs). It also helps to control the intracellular trafficking of RNPs. These roles are mediated primarily via protein–protein interactions with viral and possibly cellular proteins. Here, the regions of M1 involved in binding the viral RNPs and in mediating homo-oligomerization are identified. In vitro, by using recombinant proteins, it was found that the middle domain of M1 was responsible for binding NP and that this interaction did not require RNA. Similarly, only M1 polypeptides containing the middle domain were able to bind to RNP–M1 complexes isolated from purified virus. When M1 self-association was examined, all three domains of the protein participated in homo-oligomerization although, again, the middle domain was dominant and self-associated efficiently in the absence of the N- and C-terminal domains. However, when the individual fragments of M1 were tagged with green fluorescent protein and expressed in virus-infected cells, microscopy of filamentous particles showed that only full-length M1 was incorporated into budding virions. It is concluded that the middle domain of M1 is primarily responsible for binding NP and self-association, but that additional interactions are required for efficient incorporation of M1 into virus particles

Light does not degrade the constitutively expressed BMAL1 protein in the mouse suprachiasmatic nucleus

Biological rhythms in mammals are driven by a central circadian clock located in the suprachiasmatic nucleus (SCN). At the molecular level the biological clock is based on the rhythmic expression of clock genes. Two basic helix-loop-helix (bHLH)/PAS-containing transcription factors, CLOCK and BMAL1 (MOP3), provide the basic drive to the system by activating transcription of negative regulators through E box enhancer elements. A critical feature of circadian timing is the ability of the clockwork to be entrained to the environmental light/dark cycle. The light-resetting mechanism of the mammalian circadian clock is poorly understood. Light-induced phase shifts are correlated with the induction of the clock genes mPer1 and mPer2 and a subsequent increase in mPER1 protein levels. It has previously been suggested that rapid degradation of BMAL1 protein in the rat SCN is part of the resetting mechanism of the central pacemaker. Our study shows that BMAL1 and CLOCK proteins are continuously expressed at high levels in the mouse SCN, supporting the hypothesis that rhythmic negative feedback plays the major role in rhythm generation in the mammalian pacemaker. Using both immunocytochemistry and immunoblot analysis, our studies demonstrate that BMAL1 protein in the mouse SCN is not affected by a phase-resetting light pulse. These results indicate that rapid degradation of BMAL1 protein is not a consistent feature of resetting mechanisms in rodents

A clock shock: mouse CLOCK is not required for circadian oscillator function

The circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function

Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions ▿

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner

Casein Kinase 1 Delta Regulates the Pace of the Mammalian Circadian Clock ▿ †

Both casein kinase 1 delta (CK1δ) and epsilon (CK1ɛ) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1δ and CK1ɛ in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1δΔ2). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1δ. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by ∼20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1δ-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1ɛ did not alter these circadian endpoints. These results reveal important functional differences between CK1δ and CK1ɛ: CK1δ plays an unexpectedly important role in maintaining the 24-h circadian cycle length