Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA–mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.

The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separaseindependent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.Three essential SMC (structural maintenance of chromosomes) complexes control chromosome dynamics: condensin, cohesin, and the Smc5-Smc6 complex (37). They are composed of SMC heterodimers: Smc2 and -4 in condensin, Smc1 and -3 in cohesin, and Smc5 and -6 in Smc5-Smc6. These are large ATPases with globular N and C termini, which are separated by long coiled-coil domains. The termini interact through an ABC-like coordination of ATP through Walker A and B motifs, with the coiled-coils bending at a flexible "hinge" that acts as the SMC dimerization domain. Each complex contains a number of unique non-Smc subunits, which are likely to contribute to its unique function. Among these is a kleisin subunit, which interacts with both the SMC subunits, closing a potential ring-shaped structure (55, 61).Condensin is localized to chromosomes primarily during mitosis and is essential for mitotic chromosome condensation. Conversely, cohesin is localized primarily to interphase chromosomes and has been postulated to form a ring-shaped structure around sister chromatids to ensure their cohesion, which is important for DNA repair by homologous recombination (HR). As its name suggests, the function of the Smc5-Smc6 complex is relatively poorly understood.Scc2/4 loads cohesin onto chromosomes in G 1 , and sister chromatid cohesion is established during replication via the action of the acetyltransferase Eco1. Cohesin must be removed before chromosome segregation, where cleavage of the kleisin subunit Scc1 by the protease Separase is critical (51). In Saccharomyces cerevisiae, Separase-mediated Scc1 cleavage is essential for the removal of cohesin from all loci. In mammals, most cohesin is removed from chromosome arms early in mitosis in a Separase-independent process regulated by cohesin phosphorylation (28, 76). At anaphase, Separase-dependent removal of cohesin at the kinetochores ensures sister chromatid separation. In Schizosaccharomyces pom...

We describe two RING finger proteins in the fission yeast Schizosaccharomyces pombe, Rfp1 and Rfp2. We show that these proteins function redundantly in DNA repair. Rfp1 was isolated as a Chk1-interacting protein in a two-hybrid screen and has high amino acid sequence similarity to Rfp2. Deletion of either gene does not cause a phenotype, but a double deletion (rfp1⌬rfp2⌬) showed poor viability and defects in cell cycle progression. These cells are also sensitive to DNA-damaging agents, although they maintained normal checkpoint signaling to Chk1. Rfp1 and Rfp2 are most closely related to human Rnf4, and we showed that Rnf4 can substitute functionally for Rfp1 and/or Rfp2. The double mutants also showed significantly increased levels of protein SUMOylation, and we identified an S. pombe Ulp2/Smt4 homolog that, when overexpressed, reduced SUMO levels and suppressed the DNA damage sensitivity of rfp1⌬ rfp2⌬ cells.The fidelity of cell cycle progression is paramount for tissue homeostasis and development and avoidance of disease. A major contributor to this fidelity are the plethora of responses that detect damage to DNA, ensure its repair, and activate checkpoints that modify the cell cycle to allow time for these events to occur. These responses are rapid, highly conserved, and frequently regulated by cascades of post-translational modifications that control the activities of repair proteins and signal transducers of the checkpoints.The fission yeast Schizosaccharomyces pombe is an important model system for the dissection of the molecular controls over DNA damage responses (1, 2). S. pombe has a prominent G 2 period in vegetative cell cycles that represents ϳ70% of total cell cycle time (3). The DNA damage checkpoint functioning during this period signals to activate the Chk1 protein kinase, which in turn prevents entry into mitosis through modulating the Cdc2 regulators Wee1 and Cdc25 (4 -6). Chk1 activation is controlled by reversible phosphorylation on Ser-345 by Rad3 (7-9), an ATR (ataxia telangiectasia-and Rad3-related) homolog, although the mechanism by which this activation occurs is largely obscure. ATR homologs also phosphorylate a number of other proteins in this pathway (1, 10).In addition to phosphorylation cascades, other post-translational modifications regulate DNA damage response pathways.Among these are the covalent modification of proteins by ubiquitin and its related protein, SUMO (small ubiquitin-like modifier). In addition to directing the proteolysis of its targets, ubiquitination can directly influence responses to DNA damage. A well documented example of this is through the post-replication repair proteins that control the mono-and polyubiquitination of proliferating cell nuclear antigen, which in turn enables the bypass of lesions during DNA replication (11-13).SUMO modification of several DNA repair enzymes has also been described (14), although in most cases the molecular effects of such modification remains unclear. Control over protein SUMOylation is achieved by a balance of the activi...

CUL4A and B encode subunits of E3-ubiquitin ligases implicated in diverse processes including nucleotide excision repair, regulating gene expression and controlling DNA replication fork licensing. But, the functional distinction between CUL4A and CUL4B, if any, is unclear. Recently, mutations in CUL4B were identified in humans associated with mental retardation, relative macrocephaly, tremor and a peripheral neuropathy. Cells from these patients offer a unique system to help define at the molecular level the consequences of defective CUL4B specifically. We show that these patient-derived cells exhibit sensitivity to camptothecin (CPT), impaired CPT-induced topoisomerase I (Topo I) degradation and ubiquitination, thereby suggesting Topo I to be a novel Cul4-dependent substrate. Consistent with this, we also find that these cells exhibit increased levels of CPT-induced DNA breaks. Furthermore, over-expression of known CUL4-dependent substrates including Cdt1 and p21 appear to be a feature of these patient-derived cells. Collectively, our findings highlight the interplay between CUL4A and CUL4B and provide insight into the pathogenesis of CUL4B-deficiency in humans.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases a, d, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 3 10 3 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases d and ε. The DNA polymerase a-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase a-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase a.