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PubMed2002

Nek11, a new member of the NIMA family of kinases, involved in DNA replication and genotoxic stress responses.

DNA replication and genotoxic stresses activate various checkpoint-associated protein kinases, and checkpoint dysfunction often leads to cell lethality. Here, we have identified new members of the mammalian NIMA family of kinases, termed Nek11L and Nek11S (NIMA-related kinase 11 Long and Short isoform) as novel DNA replication/damage stresses-responsive kinases. Molecular cloning and biochemical studies showed that the catalytic domain of Nek11 is most similar to Nek4 and Nek3, and substrate specificity of Nek11L is distinguishable from those of NIMA and Nek2. The expression of nek11L mRNA increased through S to G(2)/M phase, and subcellular localization of Nek11 protein altered between interphase and prometaphase, suggesting multiple roles of Nek11. We found an activation of Nek11 kinase activity when cells were treated with various DNA-damaging agents and replication inhibitors, and this activation of Nek11 was suppressed by caffeine in HeLaS3 cells. The transient expression of wild-type Nek11L enhanced the aphidicolin-induced S-phase arrest, whereas the aphidicolin-induced S-phase arrest was reduced in the U2OS cell lines expressing kinase-negative Nek11L (K61R), and these cells were more sensitive to aphidicolin-induced cell lethality. Collectively, these results suggest that Nek11 has a role in the S-phase checkpoint downstream of the caffeine-sensitive pathway.

Amino Acid Sequence↗
PubMed1978

DNA replication pattern and cell wall growth in Escherichia coli PAT 84.

An electron microscopic radioautographic study was made of tritiated thymidine incorporation into the genome of Escherichia coli PAT 84 and of tritiated meso-D,L-2,6-diaminopimelic acid (DAP) into the cell envelope. Pulse-labeled cells growing at 30 degrees C with a doubling time of 170 min were classified according to length by the method of agar filtration. Mathematical analysis of the length distribution led to the assumption of an exponential relation between length and time. A novel DNA replication pattern was found. Within the cell cycle DNA replication terminates at 70 min; then a gap follows of 64 min, after which DNA replication is initiated at 134 min. Thus, the C period is 106 min and the D period is 100 min. Cell constriction starts at 141 min and coincides with initiation of DNA replication. Detailed quantitative analysis of the [3H]thymidine grain frequency distribution allowed the distinction of three groups of cells. The first group incorporated no label, the second group an amount C, and the third group an amount 2 X C. The relative contribution of each group to a particular length class was determined. The data fitted very well into the DNA replication pattern. The same analysis was carried out on DAP pulse-labeled cells. Again, three groups of cells could be distinguished, and their relative contributions to each length class was determined. The group with the double amount of label was especially prominent at the end of the cell cycle. The emergence of this group might represent the acquisition of new lateral growth areas.

Cell Cycle↗
PubMed2000

n-Butyrate mediated inhibition of papovavirus DNA replication in vivo and in cell culture: a mechanistic approach.

n-Butyrate, an inhibitor of G1-to-S transition inhibits papovavirus DNA replication in cell culture. To explore the efficacy of n-butyrate in vivo and to better understand its mechanism, we studied the effect of n-butyrate on viral DNA replication in mice acutely infected with polyomavirus and in the papovavirus-infected cells in culture. Newborn mice treated with n-butyrate stop growing and become runted. When infected with polyomavirus, these mice show a strong overall inhibition of viral DNA. However, a notable exception to this was the continued viral DNA replication in the differentiated mouse keratinocytes and renal epithelial cells as determined by in situ hybridization. n-Butyrate significantly inhibited viral DNA replication in the cultured IDL cells, and in polyomavirus-infected C2C12 myoblasts based on Southern blot analysis and in situ hybridization. DNA polymerase alpha (but not DNA polymerase beta) and the characteristic nuclear expression of PCNA were both inhibited in the n-butyrate treated IDL and C2C12 cells. n-Butyrate, therefore, inhibited host and viral DNA synthesis in the undifferentiated cells.

Animals↗
PubMed1994

Evolutionary role of abortive transcript as a primer for DNA replication.

Abortive cycling features transcription initiation by RNA polymerase in both prokaryote and eukaryote. It is known that T7 RNA polymerase produces abortive transcripts up to eight ribonucleotides in length depending on the initial sequence of the DNA message. On the other hand, T7 RNA polymerase initiates DNA replication from the T7 primary origin by synthesizing primers. And the shortest primer from the phi l.lB promoter in the primary origin also seems to be eight ribonucleotides in length. Therefore, it is likely that the longest abortive transcript serves as the shortest primer for T7 DNA replication from the primary origin. Considering that promoters often exist in DNA replication origins for example, E. coli oriC and many eukaryotic origins, the early DNA replication system appears to have taken advantage of the abortive cycling of RNA-dependent RNA polymerase that already existed before the emergence of DNA world. The evolutionary primitive RNA polymerase could do both transcription and priming of DNA replication. Accordingly, abortive cycling would play an important role in evolution at the emergence of DNA world. The priming activity of the primitive RNA polymerase would be taken over by primase later, which seems to be a specialized RNA polymerase for abortive cycling.

Biological Evolution↗
PubMed1996

Evidence for activities inhibiting in trans initiation of DNA replication in extract prepared from irradiated cells.

We have previously shown that replication in vitro of plasmids containing the Simian virus 40 (SV40) origin of replication is reduced when an extract of irradiated cells is used (Wang et al., Radiat. Res. 142, 169-175, 1995). We proposed that the observed reduction in the overall replication activity is due to a reduction in the efficiency of initiation events, and that it is caused by the induction or activation by ionizing radiation of a factor(s) that inhibits DNA replication in trans. Here, we extend these studies and provide evidence that the reduced replication activity of an extract prepared from irradiated cells is not the result of a nonspecific inactivation of proteins or of an increase in the requirement for SV40 large tumor antigen (TAg), the only noncellular protein required for in vitro DNA replication. Mixing experiments demonstrate the presence of a dominant inhibitory activity(ies) in the extract of irradiated cells that efficiently stalls replication in reactions assembled using extract of nonirradiated cells. The inhibitory activity is a stable, nondialyzable molecule. Studies of kinetics suggest that the inhibitory activity(ies) affects the initiation steps of DNA replication and acts, at least partly, by modifying TAg, the key initiation protein of SV40 ori DNA replication. It is likely that the same inhibitory activity(ies) regulates cellular DNA replication by modifying the cellular homologues of TAg. Purification and characterization of this inhibitory activity(ies) will contribute to our understanding of the mechanism developed by the cell to regulate DNA replication after exposure to ionizing radiation and will define a checkpoint operating in S phase. Genetic evidence for a checkpoint in S phase distinct from the checkpoints operating in G1 and G2 phase has been reported in yeast.

Antigens, Polyomavirus Transforming↗
PubMed2005

Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication.

Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.

Bacillus subtilis↗
PubMed2000

Polyomavirus large T antigen mutants affected in viral DNA replication.

We have characterized two polyomavirus large T antigen mutants with different properties in viral DNA replication. dl-97, a mutant active in immortalization, exerts a dominant negative effect in viral DNA replication. 13val, which is defective in both immortalization and viral DNA replication, has a lesion in the putative DnaJ domain affecting the block of Rb function.

3T3 Cells↗
PubMed1990

Effect of linoleic acid hydroperoxide on adenovirus DNA replication.

The effect of linoleic acid hydroperoxide on replication of adenovirus type 2 DNA in HeLa cells was examined. The level of the DNA was extremely reduced when the cells immediately after the infection with adenovirus type 2 were incubated for 24 h in the presence of 1 nmol/ml (measured by the thiobarbituric acid reaction and expressed in terms of malondialdehyde) of the hydroperoxide. The levels of the messenger RNA for adenovirus type 2 DNA polymerase and the expressed polymerase were equally reduced. The inhibition of DNA replication by lipid hydroperoxides in animal cells was discussed in relation to degenerative disorders.

Adenoviruses, Human↗
PubMed1988

A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs.

The CTF/NF-I group of cellular DNA binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription as well as DNA replication. Molecular analysis of human CTF/NF-I complementary DNA clones reveals multiple messenger RNA species containing alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis establish that individual gene products can bind to GCCAAT recognition sites and serve both as promoter-selective transcriptional activators and as initiation factors for DNA replication.

Adenoviridae↗
PubMed1995

Participation of the human beta-globin locus control region in initiation of DNA replication.

The human beta-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human beta-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3' to 5' direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.

Animals↗
PubMed2006

DNA replication in the fission yeast: robustness in the face of uncertainty.

DNA replication, the process of duplication of a cell's genetic content, must be carried out with great precision every time the cell divides, so that genetic information is preserved. Control mechanisms must ensure that every base of the genome is replicated within the allocated time (S-phase) and only once per cell cycle, thereby safeguarding genomic integrity. In eukaryotes, replication starts from many points along the chromosome, termed origins of replication, and then proceeds continuously bidirectionally until an opposing moving fork is encountered. In contrast to bacteria, where a specific site on the genome serves as an origin in every cell division, in most eukaryotes origin selection appears highly stochastic: many potential origins exist, of which only a subset is selected to fire in any given cell, giving rise to an apparently random distribution of initiation events across the genome. Origin states change throughout the cell cycle, through the ordered formation and modification of origin-associated multisubunit protein complexes. State transitions are governed by fluctuations of cyclin-dependent kinase (CDK) activity and guards in these transitions ensure system memory. We present here DNA replication dynamics, emphasizing recent data from the fission yeast Schizosaccharomyces pombe, and discuss how robustness may be ensured in spite of (or even assisted by) system randomness.

DNA Replication↗
PubMed1997

p56(chk1) protein kinase is required for the DNA replication checkpoint at 37 degrees C in fission yeast.

Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C.

Checkpoint Kinase 1↗
PubMed2006

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

Adenosine Triphosphatases↗
PubMed2000

Effect of a dCTP:dTTP pool imbalance on DNA replication fidelity in Friend murine erythroleukemia cells.

Nucleotide pool imbalances have been reported to affect the fidelity of DNA replication and repair in prokaryotic and eukaryotic cells. We have reported previously that the mutagen-hypersensitive thymidine kinase (TK)-deficient Friend erythroleukemia (FEL) cells (subclones 707BUF and 707BUE), have a more than sixfold increase in the dCTP:dTTP pool ratio when compared to that of wild-type, TK-positive (TK(+)) clone 707 cells. In this study we present the results of an investigation of the effect of the dCTP:dTTP pool imbalance on the accuracy of DNA replication within 707BUF cells. We examined the spontaneous mutation spectra occurring at the adenine phosphoribosyltransferase (aprt) locus within clone 707 (TK(+)) and 707BUF (TK(-)) FEL cells. Mutations recovered at the aprt locus in FEL cells comprised: base substitutions (43:73), frameshifts (14:13.5), and deletions (43:13.5) [clone 707 (TK(+)):707BUF (TK(-)), respectively, expressed as percentages]. A comparison of the mutation spectra obtained for the two cell lines did not reveal any significant increase in misincorporation of dCTP, the nucleotide in excess, in 707BUF (TK(-)) cells, during DNA replication synthesis. These data suggest that the dCTP:dTTP pool imbalance does not alter the fidelity of DNA replication synthesis in 707BUF (TK(-)) FEL cells. Rather, the predominance of GC --> AT transitions (53%) in the 707BUF (TK(-)) spectrum may reflect a reduced efficiency of repair by uracil DNA glycosylase of uracil residues within these cells.

Adenine Phosphoribosyltransferase↗
PubMed2006

Surface mutagenesis of the bovine papillomavirus E1 DNA binding domain reveals residues required for multiple functions related to DNA replication.

The E1 protein from papillomaviruses is a multifunctional protein with complex functions required for the initiation of viral DNA replication. We have performed a surface mutagenesis of the well-characterized E1 DNA binding domain (DBD). We demonstrate that substitutions of multiple residues on the surface of the E1 DBD are defective for DNA replication without affecting the DNA binding activity of the protein. The defects of individual substitutions include failure to form the double trimer that melts the ori and failure to form the double hexamer that unwinds the ori. These results demonstrate that the DBD plays an essential role in multiple DNA replication-related processes apart from DNA binding.

Amino Acid Substitution↗
PubMed1983

Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.

Adenoviruses, Human↗
PubMed2006

Free energy simulations of uncatalyzed DNA replication fidelity: structure and stability of T.G and dTTP.G terminal DNA mismatches flanked by a single dangling nucleotide.

A reference system for DNA replication fidelity was studied by free energy perturbation (FEP) and linear interaction energy (LIE) methods. The studied system included a hydrated duplex DNA with the 5'-CG dangling end of the templating strand, and dCTP4-.Mg2+ or dTTP4-.Mg2+ inserted opposite the dangling G to form a correct (i.e., Watson-Crick) or incorrect (i.e., wobble) base pair, respectively. The average distance between the 3'-terminal oxygen of the primer strand and the alpha-phosphorus of dNTP was found to be 0.2 A shorter for the correct base pair than for the incorrect base pair. Binding of the incorrect dNTP was found to be disfavored by 0.4 kcal/mol relative to the correct dNTP. We estimated that improved binding and more near-attack configurations sampled by the correct base pair should translate in aqueous solution and in the absence of DNA polymerase into a six times faster rate for the incorporation of the correct dNTP into DNA. The accuracy of the calculated binding free energy difference was verified by examining the relative free energy for melting duplex DNA containing GC and GT terminal base pairs flanked by a 5' dangling C. The calculated LIE and FEP free energies of 1.7 and 1.1 kcal/mol, respectively, compared favorably with the experimental estimate of 1.4 kcal/mol obtained using the nearest neighbor parameters. To decompose the calculated free energies into additive electrostatic and van der Waals contributions and to provide a set of rigorous theoretical data for the parametrization of the LIE method, we suggested a variant of the FEP approach, for which we coined a binding-relevant free energy (BRFE) acronym. BRFE approach is characterized by its unique perturbation pathway and by its exclusion of the intramolecular energy of a rigid part of the ligand from the total potential energy.

Base Pair Mismatch↗
PubMed2001

Loss of mitochondrial DNA in rabbit bladder smooth muscle following partial outlet obstruction results from lack of organellar DNA replication.

When the rabbit bladder outlet is partially obstructed, the relative amount of mitochondrial (mt) DNA per cell in bladder smooth muscle falls rapidly. In order to assess whether this loss of organellar genome results from attenuation of mt DNA replication, we cloned portions of rabbit genes specifying the single-strand binding (SSB) protein required for initiation of mt DNA replication, and the catalytic subunit of DNA polymerase gamma (pol gamma), the replication enzyme itself. We then designed primer-probe systems for real-time RT-PCR (TaqMan) analyses for each gene. These were used to assess mRNA in preparations from bladder smooth muscle and mucosa from rabbits subjected to surgical obstruction of the bladder outlet for up to 14 days. mRNA from the pol gamma gene remained essentially at control level in smooth muscle and mucosa in all samples. In mucosa, mRNA from the SSB protein gene remained virtually at control levels in all samples, as did mt genome copy number. In smooth muscle, however, levels of this mRNA declined by >95% within 3 days of obstruction and remained at that level through 14 days; this attenuation of SSB protein mRNA paralleled the loss of mt DNA in the same smooth muscle samples. Thus, lack of mt SSB protein, and consequently attenuated mt DNA replication, is a primary factor in the loss of mt genome copies in bladder smooth muscle after outlet obstruction in the rabbit model of benign bladder dysfunction.

Animals↗