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.

Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.Progression through the eukaryotic cell cycle requires the activity of a family of kinases known as cyclin-dependent kinases (cdks). cdks are inactive as monomers but become active upon heterodimerization with regulatory subunits known as cyclins. The assembly of these cyclin-cdk complexes is further regulated by the temporal expression of different cyclins so that only certain cyclin-cdk complexes are present during a given phase of the cell cycle. In early G 1 phase of the cell cycle, cyclin D is complexed with Cdk4 or Cdk6 and phosphorylates the retinoblastoma protein (pRb), an early event in the G 1 -to-S transition. This is followed by the activation of cyclin E-cdk2 (late G 1 ) and then cyclin A-cdk2 (S phase), which are responsible for the initiation of DNA replication and progression through S phase. In G 2 phase of the cell cycle, cyclin B-cdc2 begins to accumulate and drives cells through mitosis, at which time the cell cycle is allowed to begin again. These topics have been extensively reviewed (16,17,20).In addition to the temporal control of cyclin-cdk complexes, which restricts their activity to distinct periods in the cell cycle, cells have devised mechanisms for targeting cdks to specific proteins during their window of activity. We first noted this targeting mechanism in structure-function studies of p21; two independent motifs were identified, either of which could target p21 to cyclins and at least one of which was essential for optimal inhibition of kinase activity by p21 (5). ...

PurposeMutations in RHO, the gene for a rhodopsin, are a leading cause of autosomal dominant retinitis pigmentosa. The objective of this study was to determine if a synthetic retinal analogue (SRD005825) serves as a pharmacologic chaperone to promote appropriate membrane trafficking of a mutant version of human rhodopsin.MethodsA tetracycline-inducible cell line was used to produce human wild-type and T17M opsin. A cell-free assay was used to study the impact of SRD005825 on binding of 9-cis-retinal to wild-type opsin. A cell-based assay was used to measure the effect of SRD005825 on the generation of rhodopsin by spectroscopy and Western blot and the transport of rhodopsin to the cell membrane by confocal microscopy. Mice bearing T17M RHO were treated with daily oral doses of SRD005825, and retinal degeneration was measured by spectral-domain optical coherence tomography and, at the conclusion of the experiment, by electroretinography and morphometry.ResultsSRD005825 competed with 9-cis-retinal for binding to wild-type opsin but promoted the formation of rhodopsin in HEK293 cells and the trafficking of T17M rhodopsin to the plasma membrane of these cells. T17M transgenic mice exhibited rapid retinal degeneration, but thinning of the outer nuclear layer representative of photoreceptor cell bodies was delayed by treatment with SRD005825. Electroretinography a-wave and b-wave amplitudes were significantly improved by drug treatment.ConclusionsSRD005825 promoted the reconstitution of mutant rhodopsin and its membrane localization. Because it delayed retinal degeneration in the mouse model, it has potential as a therapeutic for autosomal dominant retinitis pigmentosa.Translational RelevanceSRD005825 may be useful as a treatment to delay retinal degeneration in retinitis pigmentosa patients with rhodopsin mutations causing misfolding of the protein.