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PubMed1996

Transcriptional terminators of RNA polymerase II are associated with yeast replication origins.

The compact organization of the Saccharomyces cerevisiae genome necessitates that non-coding regulatory sequences reside in close proximity to one another. Here we show there is an intimate association between transcription terminators and DNA replication origins. Four replication origins were analyzed in a reporter gene assay that detects sequences that direct 3' end formation of mRNA transcripts. All four replication origins function as orientation-independent transcription terminators in this system, producing truncated polyadenylated mRNAs. Despite this close association, the cis-acting elements that confer replication origin function are genetically separable from those required for transcription termination. Several models are explored in an attempt to address how and why the signals specifying transcription termination and replication initiation overlap.

Base Sequence↗

PubMed1996

Utilization of the same DNA replication origin by human cells of different derivation.

In the past, a highly sensitive and efficient method was developed to map DNA replication origins in human cells, based on quantitative PCR performed on nascent DNA samples. This method allowed the identification of a replication origin in the myeloid HL-60 cell line, located on chromosome 19 within an approximately 500 bp segment near the lamin B2 gene [Giacca et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 7119]. The same procedure has now been further simplified and extended to a variety of other exponentially growing human cells of different histological derivation (three neural, one connectival and one epithelial), with a nearly diploid chromosomal content. In all the six cell lines tested, the origin activity within the lamin B2 gene domain was localized to the same region. Furthermore, the lamin B2 origin was also found to be active in stimulated, but not in quiescent, peripheral blood lymphocytes.

Bone Marrow Cells↗

PubMed1997

An Abf1p C-terminal region lacking transcriptional activation potential stimulates a yeast origin of replication.

Although it has been demonstrated that eukaryotic cellular origins of DNA replication may harbor stimulatory elements that bind transcription factors, how these factors stimulate origin function is unknown. In Saccharomyces cerevisiae , the transcription factor Abf1p stimulates origin function of ARS121 and ARS1 . In the results presented here, an analysis of Abf1p function has been carried out utilizing LexA(BD)-Abf1p fusion proteins and an ARS 121 derivative harboring LexA DNA-binding sites. A minimal region which stimulates origin function mapped to 50 amino acids within the C-terminus of Abf1p. When tested for transcriptional activation of a LacZ reporter gene, the same LexA(BD)-Abf1p fusion protein had negligible transcriptional activation potential. Therefore, stimulation of ARS 121 may occur independently of a transcriptional activation domain. It has been previously observed that the Gal4p, Rap1p DNA-binding sites and the LexA-Gal4p fusion protein can replace the role of Abf1p in stimulating ARS 1 . Here we show that the stimulatory function of Abf1p at ARS 121 cannot be replaced by these alternative DNA-binding sites and the potent chimeric transcriptional activator LexA(BD)-Gal4(AD)p . Hence, these results strongly suggest that the Abf1p stimulation of replication may differ for ARS 121 and ARS 1 , and imply specificity in the Abf1p/ARS 121 relationship.

Bacterial Proteins↗

PubMed1999

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Animals↗

PubMed2002

Human Mcm proteins at a replication origin during the G1 to S phase transition.

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p-Orc6p). While the order of assembly--Mcm binding follows ORC binding--seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G1 phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.

Blotting, Western↗

PubMed2002

Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins.

Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.

Animals↗

PubMed2001

Value of FDG PET in patients with fever of unknown origin.

Fever of unknown origin (FUO) is a diagnostic challenge, because the cause of such fever may be manifold. Studies on the use of positron emission tomography (PET) using 18F-fluorodeoxyglucose (18F-FDG), for the diagnosis of inflammation in patients with osteomyelitis or HIV have been promising and suggest its use in patients with FUO. In this study, we used FDG PET in 16 patients with FUO in whom conventional diagnostics had not been conclusive. In 12 patients, (75%) non-physiological accumulations of FDG were found which led to the final diagnosis in 11 patients (69%). FDG PET was negative in four patients (25%). Two of these patients had rheumatic fever, while in the other two patients the origin of fever could not be detected within 3 months after PET by any other laboratory or imaging means. These findings point to the high sensitivity of FDG whole-body PET for the detection of morphologically assessable foci as an origin of FUO. Moreover, they suggest a high negative predictive value of FDG PET in the setting of FUO, since in no patient with a negative FDG PET could a morphological origin of the fever be determined. In conclusion, FDG whole-body PET appears to be a promising diagnostic tool in patients with FUO, in whom conventional diagnostics had been unsuccessful.

Adult↗

PubMed2002

Noisy clues to the origin of life.

The origin of stable self-replicating molecules represents a fundamental obstacle to the origin of life. The low fidelity of primordial replicators places restrictions on the quantity of information encoded in a primitive nucleic acid alphabet. Further difficulties for the origin of life are the role of drift in small primordial populations, reducing the rate of fixation of superior replicators, and the hostile conditions increasing developmental noise. Thus, mutation, noise and drift are three different stochastic effects that are assumed to make the evolution of life improbable. Here we show, to the contrary, how noise present in hostile early environments can increase the probability of faithful replication, by amplifying selection in finite populations. Noise has negative consequences in infinite populations, whereas in finite populations, we observe a synergistic interaction among noise sources. Hence, two factors formerly considered inimical to the origin of life-developmental noise and drift in small populations-can in combination give rise to conditions favourable to robust replication.

Biological Evolution↗

PubMed1997

Stringent structural and sequence requirements of the human herpesvirus 6B lytic-phase origin of DNA replication.

The lytic-phase origin of DNA replication from human herpesvirus 6B (HHV-6B oriLyt) contains two binding sites for the origin-binding protein (OBPH6B), both of which are required for DNA replication and which are separated by an AT-rich spacer. We have tested the functional significance of the structural, spatial and sequence characteristics of this spacer element by constructing a series of mutated origin sequences and analysing their replication efficiency. Changes in the sequence composition of length of the spacer resulted in dramatic decreases in replication efficiency. Furthermore, in contrast to what has been observed for herpes simplex virus type 1 (HSV-1) oriS, insertion of a complete helical turn of DNA into the spacer also resulted in abrogation of origin function. These data suggest that the arrangement of OBP sites in HHV-6B oriLyt is stringently constrained in terms of spacing and intervening sequence.

Base Sequence↗

PubMed1998

Active domains of human papillomavirus type 11 E1 protein for origin replication.

Viral proteins E1 and E2 are essential for transient human papillomavirus (HPV) DNA replication. E1 is a multifunctional protein which can bind DNA and complex with E2, has ATPase and helicase activities, and interacts with DNA polymerase alpha-primase. E2 is a transactivator-repressor protein, playing an important role in replication and transcriptional regulation. A series of deletion mutants of HPV-11 E1 were constructed and tested in functional assays to define those domains of HPV-11 E1 which are important for binding to the origin DNA and E2. The domain of HPV-11 E1 involved in binding to the origin was located between aa 186 and 649, and that for binding to E2 was between aa 346 and 649. Since E1 binds to the origin more efficiently in the presence of E2, we also mapped the DNA binding domain of E1 in the presence of E2, and found that when binding was enhanced, the region of E1 involved in binding was similar to that observed with E1 alone. The same deletion mutation constructs of E1 were subcloned into an expression vector for use in transient replication assays to study the effect of the deletions on the replication of the origin DNA in vivo and the data suggest that the C-terminal domain contains important functions for replication.

Binding Sites↗

PubMed2003

Sequence-independent DNA binding and replication initiation by the human origin recognition complex.

We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.

Animals↗

PubMed2002

Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus.

How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of approximately 150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing in sir2 Delta mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.

DNA Replication↗

PubMed1994

Activation of a yeast replication origin near a double-stranded DNA break.

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

DNA Damage↗

PubMed2001

Functionally distinct, sequence-specific replicator and origin elements are required for Drosophila chorion gene amplification.

To meet the demand for the rapid synthesis of chorion (eggshell) proteins, Drosophila ovarian follicle cells amplify the chromosomal loci containing the chorion gene clusters up to 60-fold. Amplification occurs by repeated firing of one or more origins located within each gene cluster. Deletion analyses of transgenic constructs derived from the third chromosome cluster have identified a 320-bp amplification control element (ACE3) required for amplification, as well as several stimulatory amplification enhancing regions (AERs). Two-dimensional (2D) gel analyses have identified multiple DNA replication initiation sites (origins) that partially overlap in location with ACE3 and the AERs. To further study sequence requirements for amplification, a vector was used in which transgenic constructs are protected from chromosomal position effects by flanking insulator elements, the suppressor Hairy-wing protein binding site (SHWBS). Using the buffered vector, the 320-bp ACE3 and an 884-bp element designated ori-beta were found to be necessary and sufficient for amplification. Two-dimensional gels revealed that ori-beta was acting as the origin. In contrast, origin activity could not be detected for ACE3. An insulator placed between ACE3 and ori-beta inhibited amplification, indicating that ACE3 activates ori-beta in cis. The results suggest that ACE3 acts as a replicator and support and extend the replicator model for the organization of metazoan chromosomal replicons.

Animals↗

PubMed2000

Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids.

The Lyme disease agent Borrelia burgdorferi has a genome composed of a linear chromosome and a series of linear and circular plasmids. We previously mapped the oriC of the linear chromosome to the center of the molecule, where a pronounced switch in CG skew occurs. In this study, we analyzed B. burgdorferi plasmid sequences for AT and CG skew in an effort to similarly identify plasmid replication origins. Cumulative skew diagrams of the plasmids suggested that they, like the linear chromosome, replicate bidirectionally from an internal origin. The B. burgdorferi linear chromosome contains homologs to partitioning protein genes soj and spoOJ, which are closely linked to oriC at the minimum cumulative skew point of the 1-Mb molecule. A soj/parA homolog also maps to cumulative skew minima of the B. burgdorferi linear and circular plasmids, further suggesting that these regions contain the replication origin. The heterogeneity in these genes and in the nucleotide sequences of the putative origin regions could account for the mutual compatibility of the multiple DNA elements in B. burgdorferi.

Base Composition↗

PubMed2006

Nuclear transcription is essential for specification of mammalian replication origins.

I have demonstrated that nuclear transcription modulates the distribution of replication origins along mammalian chromosomes. Chinese Hamster Ovary (CHO) cells were exposed to transcription inhibitors in early G1 phase and replication origin sites in the dihydrofolate reductase (DHFR) gene locus were mapped several hours later. DNA within nuclei prepared from control and transcription-deficient G1-phase cells was replicated with similar efficiencies when introduced into Xenopus egg extracts. Replication initiated in the intergenic region within control late-G1 nuclei, but randomly within transcriptionally repressed nuclei. Random initiation was not a consequence of inability to produce an essential protein(s), since initiation was site-specific within cells exposed to the translation inhibitor cycloheximide during the same interval of G1 phase. Furthermore, in vivo inhibition of transcription within late-G1-phase cells reduced the frequency of usage of pre-established DHFR replication origin sites. Transcription rates in the DHFR domain were very low and did not change throughout G1 phase. This implies that, although ongoing nuclear transcription is required, local expression of the genes in the DHFR locus alone is not sufficient to create a site-specific replication initiation pattern. I conclude that epigenetic factors, including general nuclear transcription, play a role in replication origin selection in mammalian nuclei.

Animals↗

PubMed2004

Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically.

In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re-initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA-less cells. The mutant origins underwent re-initiation, showing that the GATC sites in oriC are required for sequestration. Each re-initiation eventually gave rise to a cell containing an extra nucleoid. The three-nucleoid cells displayed one asymmetrically placed FtsZ-ring and divided into a two-nucleoid cell and a one-nucleoid cell. The three nucleoid-cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.

Animals↗

PubMed2005

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin.

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.

Base Sequence↗