Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair.

BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

First the CDKs, now the DDKs.

In budding yeast, Dbf4p and Cdc7p control initiation of DNA synthesis. They form a protein kinase - Cdc7p being the catalytic subunit and Dbf4p a cyclin-like molecule that activates the kinase in late G1 phase. Dbf4p also targets Cdc7p to origins of replication, where probable substrates include certain Mcm proteins. Recent studies have identified Dbf4p- and Cdc7p-related proteins in fission yeast and metazoans. These homologues also phosphorylate Mcm proteins and could have a similar function to that of Dbf4p-Cdc7p in budding yeast. Thus, it seems likely that, like the cyclin-dependent kinases (CDKs), the Dbf4p-Cdc7p activity is conserved in all eukaryotes.

A fission yeast gene, him1(+)/dfp1(+), encoding a regulatory subunit for Hsk1 kinase, plays essential roles in S-phase initiation as well as in S-phase checkpoint control and recovery from DNA damage.

Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G(1)/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1(+) from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1(+) is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G(1). The protein level is low at START in G(1), increases at the G(1)/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G(1)/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1(+) is identical to rad35(+). Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

A novel growth- and cell cycle-regulated protein, ASK, activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells.

A novel human protein, ASK (activator of S phase kinase), was identified on the basis of its ability to bind to human Cdc7-related kinase (huCdc7). ASK forms an active kinase complex with huCdc7 that is capable of phosphorylating MCM2 protein. ASK appears to be the major activator of huCdc7, since immunodepletion of ASK protein from the extract is accompanied by the loss of huCdc7-dependent kinase activity. Expression of ASK is regulated by growth factor stimulation, and levels oscillate through the cell cycle, reaching a peak during S phase. Concomitantly, the huCdc7-dependent kinase activity significantly increases when cells are in S phase. Furthermore, we have demonstrated that ASK serves an essential function for entry into S phase by showing that microinjection of ASK-specific antibodies into mammalian cells inhibited DNA replication. Our data show that ASK is a novel cyclin-like regulatory subunit of the huCdc7 kinase complex and that it plays a pivotal role in G1/S transition in mammalian cells.

Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast.

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30 degrees C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37 degrees C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

Roles of the G site and phi X174-type primosome assembly site in priming of leading-strand synthesis: initiation by a mobile primosome and replication-fork arrest by RepA protein bound to oriR.

Bacterial replicons often contain single-strand initiation sequences (ssi) such as a G site (a sequence recognized by a dnaG-encoded primase for the synthesis of primer RNA) and a primosome assembly site (pas) near the DNA replication origin (ori). The R1 plasmid contains a G site downstream from oriR, which serves for the priming of the leading-strand synthesis of this plasmid. On the other hand, the F, R6K and Rts1 plasmids carry pas at similar locations relative to the respective ori. In order to assess the functional significance of these pas, R1 plasmid derivatives carrying an n'-pas (phi X174-type pas) in place of the G site were constructed and their replication properties were examined in vitro. Deletion of the G site in the R1 plasmid resulted in a nearly 80% reduction of total DNA synthesis in vitro, which was recovered to the wild-type (wt) level by inserting the G4 complementary ori. Furthermore, insertion of an n'-pas on the leading-strand template restored the in vitro replicative activity to a level 70% of wt. This recovery was dependent on the assembly of the phi X174-type primosome, which efficiently primed leading-strand synthesis and moved toward the oriR. However, the R1 plasmid derivative containing the n'-pas replicated unidirectionally in vitro, probably due to the anti-helicase activity of the RepA protein bound to oriR, which was shown by helicase assays using partial heteroduplexes as substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

A fusion protein library: an improved method for rapid screening and characterization of DNA binding or interacting proteins.

A rapid method for screening and characterization of DNA binding or protein-interacting molecules is described. The method relies on a fusion protein library in which randomized DNA fragments are inserted into pGEX-3X and its derivatives to generate collections of GST-fusion proteins. After inducing the expression of the fusion proteins by addition of IPTG, the colonies can be screened either with radioactively labeled DNA/RNA fragment for specific clones encoding DNA/RNA binding proteins or with an antibody for clones encoding proteins of interest. They can also be screened with a radioactively labeled protein for cloning of interacting molecules. The fusion proteins encoded by the isolated clones can be readily purified by conducting the lysis of the cells and an affinity column in the presence of an alkyl anionic detergent, N-laurylsarcosine (sarkosyl), and can be further characterized.

Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids.

Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.

Central gustatory paths in the crucian carp, Carassius carassius.

The central fiber connections of the gustatory system (VII, IX, and X nerves) in th crucian carp were examined by the Fink-Heimer method and its modification. The sensory and recurrence roots of the VII enter th brainstem separately and terminate in teh ipsilateral half of the facial lobe (L-VII). Afferent fibers of the IX terminate in the glossopharyngeal lobe (L-IX). Most afferent fibers of the X terminate in the sensory layer of the vagal lobe (L-X), in which degenerating terminals occur in some laminae. Some vagal afferents project bilaterally to the commissural nucleus of Cajal. The cutaneous component of the X projects to the nucleus of the spinal trigeminal tract (SpV) and the medial funicular nucleus (nFM). Ascending secondary fibers from the L-VII project bilaterally to the secondary gustatory nucleus (nGS) in the isthmus region. Descending secondary fibers from the L-VII turn caudally in the SpV. These fibers terminate mostly in the nucleus of the SpV and sparsely in the nFM. The L-IX and L-X give rise to the long and short secondary paths. The long path projects as the ascending secondary tract to the ipsilateral nGs. The short path includes secondary fibers projecting to the motor layer of the L-X and the medullary reticular formation. Teritary gustatory fibers arisig in the nGs project ipsilaterally to two diencephalic nuclei; the nucleus glomerulosus and the nucleus diffusus lobi inferioris.

Cytoarchitecture and visual receptive neurons in the Wulst of the Japanese quail (Coturnix coturnix japonica).

The cellular organization of the Wulst was studied in Nissl- and Golgi-stained brain sections in order to identify the visual receptive neurons. Golgi-impregnated neurons were divided into four types according to their soma size, dendritic configuration, and density of spine distribution. Type I neurons, the largest cells in the Wulst, have long, straight dendrites with many spines. Type II neurons are medium-sized cells with long, straight dendrites. These dendrites have numerous spines. Type III neurons are medium-sized or small cells with spine-free dendrites. Type IV neurons, the smallest cells in the Wulst, have short dendrites with sparse spines. The projections of the nucleus dorsolateralis anterior thalami pars lateralis (DLL) to the Wulst were determined by the Fink-Heimer method. After lesions of the DLL, degenerating terminals are seen in a dorsolateral portion of the nucleus intercalatus hyperstriatum accessorium where the types II, III, and IV neurons are distributed. Postsynaptic elements to the DLL axons were identified by reconstruction of electron microscopic serial sections. Most of the postsynaptic elements were dendritic spines of the type II and IV neurons and a few were dendritic shafts of the type III neurons.

Mechanisms of primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli.

In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These 'primers' are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on primer RNA synthesis on single-stranded DNA templates containing specific 'priming signals' revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a sigma 70 subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicons, some of which function in initiation of the leading strand synthesis. The latter, on the other hands involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes primer RNAs at multiple sites. Two distinct primosomes, DnaA-dependent and PriA-dependent, have been identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNaseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates primer RNA synthesis and replication fork progression.

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: involvement of ATPase/helicase activity of PriA for inducible stable DNA replication.

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the phiX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the phiX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the phiX174-type primosome at a single-stranded n'-pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.

DnaA- and PriA-dependent primosomes: two distinct replication complexes for replication of Escherichia coli chromosome.

Enzymatic analyses of primosome assembly at chromosomal and plasmid origins as well as that at single-stranded replication origins revealed the presence of two distinct primosomes in Escherichia coli for primer RNA synthesis and duplex unwinding. A DnaA-dependent primosome is assembled at oriC, the chromosomal origin of Escherichia coli, as well as at the A site, a single-stranded DNA hairpin containing a dnaA box sequence within its stem. In contrast, PriA protein recognizes a hairpin, called n'-pas (primosome assembly site), and initiates assembly of the phiX174-type PriA-dependent primosome in conjunction with other prepriming proteins. Genetic analyses of the prepriming proteins required specifically for the latter primosome strongly suggested that it is responsible for RecA-dependent, DnaA/oriC-independent replication of the Escherichia coli chromosome. Furthermore, primosome assembly in replication of various plasmids may also be classified into either DnaA-dependent or PriA-dependent type. We propose that Escherichia coli possesses two distinct, mutually exclusive primosomes which are differentially utilized by the chromosome as well as by the plasmids. PriA protein appears to be conserved in a wide range of prokaryotic species, and we will also discuss possible biological function of the PriA-dependent primosome in the process of responses to DNA damages.

CDC7 kinase complex as a molecular switch for DNA replication.

Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast Saccharomyces cerevisiae, are widely conserved in eukaryotes including fission yeast and human. Dbf4-related activators bind and stimulate kinase activity of Cdc7-like catalytic subunit. Its kinase activity is cell cycle-regulated, mainly through availability of the activation subunit whose level increases at G1/S boundary and is maintained at a high level throughout S phase. MCM2 protein is among physiologically important substrates. Genetic studies in fission yeast indicate that Cdc7-related kinase complex also functions in meiosis, uninduced mutagenesis, DNA replication checkpoint signaling and maintenance of chromatin structures during S phase.

Decreased synaptic areas on Purkinje spines in the cerebellar cortex of the mutant Japanese quail, Coturnix coturnix japonica.

'Dark frayed feather nervous disorder' (dn) is a neurological mutation in quails in which the cerebellar cortex is abnormally organized. Purkinje cells are not aligned in a single row and show hypoplasia of the dendrites. The synapses between the parallel fibers and the spines of the Purkinje cell dendrites were examined with the technique of serial sections in electron microscopy. The postsynaptic thickenings were obviously decreased in the mutant quail despite the same density and size of dendritic spines of Purkinje cells. In addition, ectopic spines and postsynaptic differentiations free of parallel fibers were not found on the dn Purkinje cell. Because of the poor dendritic arborization, the total number of spines and the total synaptic area are, therefore, reduced in the dn Purkinje cell. According to the results obtained the dn mutant genetic locus is considered to affect primarily Purkinje cells.

Inhibition of NMDA receptors and nitric oxide synthase reduces ischemic injury of the retina.

This study was performed to examine the roles of body temperature, NMDA receptors and nitric oxide (NO) synthase in post-ischemic retinal injury in rats. Cell loss in the ganglion cell layer and thinning of the inner plexiform layer were observed 7 days after ischemia. Cell loss in the ganglion cell layer but not thinning of the inner plexiform layer was reduced by hypothermia during ischemia. Intravenous injection of dizocilpine (MK-801) or Nomega-nitro-L-arginine methyl ester (L-NAME) prior to ischemia ameliorated retinal injury. These results suggest that activation of NO synthase following NMDA receptor stimulation is involved in ischemia-induced retinal injury.

Neuro-Behçet disease presenting with internuclear ophthalmoplegia.

PURPOSE: To report a case of isolated internuclear ophthalmoplegia in a patient with neuro-Behçet's disease. METHOD: We evaluated the patient's clinical course. RESULTS: The patient had isolated internuclear ophthalmoplegia and headache. Subsequent cerebrospinal fluid study revealed marked pleocytosis predominated by lymphocytes (61%) and polymorphonuclear cells (35%), increased protein content, and normal glucose level. A magnetic resonance imaging study with T2-weighted image demonstrated a hyperintense area in the medial longitudinal fasciculus. CONCLUSION: Colchicine treatment of neuro-Behçet's disease caused marked improvement in the symptoms in this case, and the midbrain lesion completely disappeared after treatment.

Ipsilateral retinal projections in Japanese quail, Coturnix coturnix japonica.

Ipsilateral retinal projections were investigated in Japanese quails by means of the Fink-Heimer method after retinal extirpation, and by means of direct injection of horseradish peroxidase or cobalt ion-tophoresis into the optic nerve. Ipsilateral projections were found in the nucleus lateralis anterior thalami, nucleus dorsolateralis anterior thalami pars lateralis, nucleus geniculatus lateralis pars ventralis, nucleus lentiformis mesencephali pars magnocellularis and nucleus ectomamillaris. No ipsilateral retino-tectal projections were observed.

Prolapsing gyrus rectus as a cause of progressive optic neuropathy.

The pathogenesis of optic neuropathy caused by neurovascular compression or by similar mechanisms is unclear. Thin-slice magnetic resonance (MR) imaging was performed in 69 patients with optic neuropathy without demonstrable ophthalmological lesions (57.0 +/- 17.1 years of age) and 102 normal subjects (57.7 +/- 13.9 years of age). The MR imaging features were classified into "no compression" by the internal carotid artery (ICA), "compression" by the ICA, "no contact" with the anterior cerebral artery (ACA) or the gyrus rectus, "contact" with either or both, "compression" by the ACA, and "compression" by the gyrus rectus. The Spearman correlation coefficients were calculated between patients or controls, the MR classification, and the age, and the number of patients in each MR classification were evaluated by the chi 2 test. Five of the 69 patients with rapidly progressive symptoms were operated on via the frontotemporal approach. The MR imaging feature of "compression" by the gyrus rectus was the best predictor of optic neuropathy (Spearman correlation coefficients rho = -0.23646, p < 0.0018). This MR imaging feature was observed in 38 of 69 patients and in 32 of 102 controls (p = 0.002). Compression of the nerve by the gyrus rectus or the ACA was confirmed in all five operated cases. Decompression of the nerve was fully achieved in four of the five patients, and their symptoms have not progressed since then. Optic neuropathies due to compression by the prolapsing gyrus rectus are not well understood. Such neuropathies may be detected by MR imaging.

[Two cases of systemic lupus erythematosus presenting with disc edema].

We report two young women, 22 and 19 years old, who showed bilateral optic disc edema in the course of systemic lupus erythematosus. Lumbar puncture showed increased intracranial hypertension with no abnormal findings in the composition of the cerebrospinal fluid. Computed tomography and magnetic resonance imaging showed no abnormal findings. They were diagnosed as having rare intracranial hypertension associated with systemic lupus erythematosus. Treatment with systemic corticosteroids produced a dramatic resolution of the increased intracranial hypertension and the disc edema.