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PubMed2003

Isoform switching of Cdc6 contributes to developmental cell cycle remodeling.

Xenopus laevis early development is characterized by rapid and synchronous cleavage cycles, which consist of alternating S and M phases. At midblastula transition, zygotic transcription begins and these cleavage cycles are replaced by longer cell division cycles that include gap phases and checkpoints. Herein, we demonstrate developmentally regulated Cdc6 isoform switching that contributes to this developmental cell cycle remodeling. Cdc6 is an essential component of the eukaryotic DNA replication machine that licenses each origin to one round of DNA replication each cell division cycle. The originally characterized Xenopus Cdc6 isoform (here termed Xcdc6A) and a novel isoform (Xcdc6B) have divergent N-terminal regulatory regions and different temporal patterns of expression. Although abundant in the early embryo, Xcdc6A becomes undetectable following midblastula transition. In contrast, while Xcdc6B is present in the early embryo, it is nonfunctional, as judged by lack of chromatin binding. In somatic tissue, however, Xcdc6B binds chromatin and its inhibition blocks entry into S phase. This is the first example of developmental regulation of Cdc6, raising intriguing implications for cell cycle remodeling during embryogenesis.

Amino Acid Sequence↗

PubMed2002

A large dispersed chromosomal region required for chromosome segregation in sporulating cells of Bacillus subtilis.

The cis-acting sequences required for chromosome segregation are poorly understood in most organisms, including bacteria. Sporulating cells of Bacillus subtilis undergo an unusual asymmetric cell division during which the origin of DNA replication (oriC) region of the chromosome migrates to an extreme polar position. We have now characterized the sequences required for this migration. We show that the previously characterized soj-spo0J chromosome segregation system is not essential for chromosome movement to the cell pole, so this must be driven by an additional segregation mechanism. Observations on a large set of precisely engineered chromosomal inversions and translocations have identified a polar localization region (PLR), which lies approximately 150-300 kbp to the left of oriC. Surprisingly, oriC itself has no involvement in this chromosome segregation system. Dissection of the PLR showed that it has internal functional redundancy, reminiscent of the large diffuse centromeres of most eukaryotic cells.

Bacillus subtilis↗

PubMed2000

Two patches of amino acids on the E2 DNA binding domain define the surface for interaction with E1.

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to the viral origin of DNA replication (ori), forming a complex which is essential for initiation of DNA replication. Cooperative binding has two components, in which (i) the DNA binding domains (DBDs) of the two proteins interact with each other and (ii) the E2 transactivation domain interacts with the helicase domain of E1. By generating specific point mutations in the DBD of E2, we have defined two patches of amino acids that are involved in the interaction with the E1 DBD. These same mutations, when introduced into the viral genome, result in severely reduced replication of the viral genome, as well as failure to transform mouse cells in tissue culture. Thus, the interaction between the E1 and E2 DBDs is important for the establishment of the viral genome as an episome and most likely contributes to the formation of a preinitiation complex on the viral ori.

Animals↗

PubMed1995

Origin of replication of the Bacillus subtilis chromosome: in vitro approach to the isolation of early replicating segments.

We have developed a permeable cell system for the study of the molecular mechanisms involved in the control and initiation of DNA replication at the origin of the Bacillus subtilis chromosome. Our system take advantage of the synchronous initiation of DNA replication that occurs in outgrowing B. subtilis spores and the curtailment of DNA elongation by novobiocin. Early replicating DNA sequences were identified by the use of 5-mercury-dCTP as substrate, which allows the isolation of nascent DNA chains by affinity chromatography on thiol agarose. The average size of the isolated nascent DNA was 1,000 bp, and more than 80% of the nascent DNA chains had RNA primers at their 5' end. The study of the temporal order of chromosome replication near the origin using this experimental system showed that a segment containing recF and gyrB replicated earlier than a segment containing gyrA and part of the rRNA operon (rrnO). This observation is in agreement with previous in vivo data on the replication of origin region and supports the conclusion that the major activity in our in vitro system was the faithful replication of the ori region.

Bacillus subtilis↗

PubMed1998

The human papillomavirus type 18 (HPV18) replication protein E1 is a transcriptional activator when interacting with HPV18 E2.

The human papillomavirus type 18 E1 and E2 proteins are both required for the initiation of viral DNA replication. Whereas E2 is the major viral transcription regulator, E1 is the replication initiator protein. They interact with each other and with the origin sequences to initiate viral DNA replication. We show that the HPV18 E1 and E2 proteins, when bound to an origin sequence cloned upstream of a heterologous promoter, synergistically activate transcription. This synergy required binding of E2 to at least two binding sites, but was partially independent of E1 binding to the origin of replication. Transcriptional activation was observed even in the absence of replication of the target DNA. Only homologous E1 and E2 proteins binding to homologous origin sequences from BPV1 or HPV18 viruses could synergistically activate transcription. We show that the HPV18 E1 protein can activate transcription when targeted to the DNA by fusion of the complete polypeptide with the BPV1 E2 C-terminus dimerization/DNA binding domain, implying that HPV18 E1 is an intrinsic transcriptional activator, though less potent than E2. The interaction between E1 and E2 may form a transcriptionally active complex during initiation of viral DNA replication.

Chloramphenicol O-Acetyltransferase↗

PubMed2001

DNA replication control through interaction of E2F-RB and the origin recognition complex.

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila dE2F1 and dDP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Here we show that mutants of Rbf (an retinoblastoma protein homologue) fail to limit DNA replication. We also show that the dDP, dE2F1 and Rbf proteins are located in a complex with DmORC, and that dE2F1 and DmORC are bound to the chorion origin of replication in vivo. Our results indicate that dE2F1 and Rbf function together at replication origins to limit DNA replication through interactions with DmORC.

Animals↗

PubMed1985

Effect of dam methylation on the activity of the E. coli replication origin, oriC.

Methylation of GATC sites by the dam methylase is required for efficient initiation of DNA replication at the replication origin, oriC, of Escherichia coli. This is demonstrated by the inability of minichromosomes to be maintained in dam mutant strains. The requirement for methylated GATC sites is less stringent in vitro than in vivo. The time required for complete methylation of the origin region apparently determines the minimal spacing of replication forks on the chromosome.

Chromosomes, Bacterial↗

PubMed1983

Regulatory sequences and virus-cell interactions of JC virus.

A set of recombinant DNAs, representing several isolates of JCV, has been constructed. Some of these cloned DNAs transform nonpermissive primary hamster brain cells. Analysis of the early viral mRNAs and proteins isolated from these transformants revealed similarities with the large and small T messages and proteins of SV40. The region of the JCV genome from 0.63 to 0.73 map units was sequenced by the Maxam-Gilbert technique. This segment of DNA specifies several regulatory elements and the amino-terminal portion of the early viral proteins. Comparisons with the analogous regions in the polyomaviruses SV40 and BKV, confirm the close evolutionary relationship of these three viruses. Similarities include palindromic and symmetrical sequences near the origins of DNA replication, binding sites for the large T proteins, and AT-rich region (the Goldberg-Hogness or TATA box), and a large tanden duplication or triplication to the late side of the replication origin (however, these sequences differ). Homology between the sequences coding for the early proteins is also evident (27 of the first 33 amino acids are shared). Of greater interest are features of the JCV genome which differ from those of other polyomaviruses. Absent in JCV and BKV are sequences which resemble the third T antigen-binding site of SV40. In addition, a set of sequences present in JCV and BKV DNA (33 nucleotides in JCV, 22 nucleotides in BKV), and located near a 17 base pair palindrome shared by all three viruses, is missing in SV40 DNA. Another sequence, GGGXGGAG, which is repeated several times in many polyomaviruses and adenoviruses, and which is thought to play a role in DNA replication and/or transcription, is not found in the JCV sequence presented. Finally, the tandem repeat of JCV, unlike those of BKV and SV40, includes the Goldberg-Hogness sequence.

Animals↗

PubMed1994

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.

Cloning, Molecular↗

PubMed2005

Epigenetic control of replication origins.

Efficient duplication of the eukaryotic genome requires the spatial and temporal coordination of numerous replication origins on each chromosome. Epigenetic factors, like chromatin environment, can have profound effects on origin site selection, utilization frequency, and cell cycle firing time. Precisely how chromatin contributes to origin site selection and timing is not completely understood. Recently, we reported on the cell cycle changes in chromatin structure at the plasmid replication origins of Epstein-Barr Virus (EBV) and Kaposi's Sarcoma-Associated Herpes virus (KSHV). These studies and others suggest that cell cycle changes in histone modification and nucleosome remodeling regulate prereplication factor assembly and initiation of DNA replication at origins. We discuss how these studies of viral origins may provide important insights into epigenetic control of cellular chromosome origins.

Epigenesis, Genetic↗

PubMed2001

Minute virus of mice initiator protein NS1 and a host KDWK family transcription factor must form a precise ternary complex with origin DNA for nicking to occur.

Parvoviral rolling hairpin replication generates palindromic genomic concatemers whose junctions are resolved to give unit-length genomes by a process involving DNA replication initiated at origins derived from each viral telomere. The left-end origin of minute virus of mice (MVM), oriL, contains binding sites for the viral initiator nickase, NS1, and parvovirus initiation factor (PIF), a member of the emerging KDWK family of transcription factors. oriL is generated as an active form, oriL(TC), and as an inactive form, oriL(GAA), which contains a single additional nucleotide inserted between the NS1 and PIF sites. Here we examined the interactions on oriL(TC) which lead to activation of NS1 by PIF. The two subunits of PIF, p79 and p96, cooperatively bind two ACGT half-sites, which can be flexibly spaced. When coexpressed from recombinant baculoviruses, the PIF subunits preferentially form heterodimers which, in the presence of ATP, show cooperative binding with NS1 on oriL, but this interaction is preferentially enhanced on oriL(TC) compared to oriL(GAA). Without ATP, NS1 is unable to bind stably to its cognate site, but PIF facilitates this interaction, rendering the NS1 binding site, but not the nick site, resistant to DNase I. Varying the spacing of the PIF half-sites shows that the distance between the NS1 binding site and the NS1-proximal half-site is critical for nickase activation, whereas the position of the distal half-site is unimportant. When expressed separately, both PIF subunits form homodimers that bind site specifically to oriL, but only complexes containing p79 activate the NS1 nickase function.

Adenosine Triphosphate↗

PubMed1994

Analysis of the DNA-binding domain of the HSV-1 origin-binding protein.

In order to understand DNA-protein interactions at the origin of DNA replication in herpes simplex virus type 1 (HSV-1), we have undertaken an analysis of the DNA-binding domain of the origin-binding protein (OBP) and its mechanism of binding to the Oris sequence of HSV-1. Mutant DNA-binding domains were constructed, expressed in vitro, and used to test for binding by gel shift analysis. A C-terminal deletion mutant was functional in binding, thereby redefining the C-terminal boundary of the DNA-binding domain at amino acid 822. Fifteen insertion mutants were also constructed across the DNA-binding domain. Several of these mutants were unable to bind DNA. Interestingly, 4 mutants that destroy DNA binding fall within a region that has a particularly high degree of sequence similarity to the varicella zoster virus gene 51 product. A second objective was to define how the DNA-binding domain interacts with the origin. Results of gel shift analysis using contranslated proteins of different sizes suggest that the DNA-binding domain can interact with a single binding site as a monomer. Binding to the wild-type Oris template indicated that the binding domains can interact with both binding sites I and II independent of any cooperative effect mediated by the amino-termini. This suggests that the basic unit of recognition involved in OBP/Oris interactions may contain a single DNA-binding domain of OBP in association with a single binding site.

Amino Acid Sequence↗

PubMed1989

Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome.

Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described. Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro. In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases. Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I. If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome. Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly. Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA. The product of this reaction, form I DNA, is more unwound than form I DNA. The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis. These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.

Bacterial Proteins↗

PubMed1989

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine.

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (Mi) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only Mi but also D varied with growth rate at generation times (tau) between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in tau, by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.

Cell Cycle↗

PubMed1982

Deletion mutants defining the Escherichia coli replication factor Y effector site sequences in pBR322 DNA.

The Escherichia coli DNA replication factor Y, along with other genetically undefined replication proteins, is involved in a dnaB-, dnaC-, and dnaG-dependent pathway of primer formation on phi X174 single-stranded circular DNA. In addition, replication factor Y has a site-specific, single-stranded DNA-dependent ATPase activity. We have previously demonstrated the presence of two factor Y effector sites on pBR322 DNA. When inserted into the filamentous phage f1R229, these sites can function as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent origins of DNA replication. We report here the construction of deletion mutants of the two pBR322 factor Y effector sites. These deleted sites no longer function as effectors for factor Y ATPase activity nor as templates for rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent DNA synthesis. We conclude that the DNA sequences required for factor Y ATPase activity and origin function are likely to be identical.

Adenosine Triphosphatases↗

PubMed2001

Replication of the rat aldolase B locus differs between aldolase B-expressing and non-expressing cells.

We previously reported a rat chromosomal origin of DNA replication (oriA1) that encompassed the aldolase B (AldB) gene promoter. Here, we examined utilization of oriA1 in AldB-expressing and non-expressing cells. The results suggested the occurrence of mutually exclusive regulation between DNA replication and transcription. Nascent strand abundance as assayed by competitive polymerase chain reaction using bromodeoxyuridine-labeled nascent DNA indicated that oriA1 is not utilized in AldB-expressing cells, while it is fired in non-expressing cells. In the latter non-expressing cells, the replication fork seemed to slow at 20-22 kb downstream of oriA1.

Animals↗

PubMed1997

Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs.

A novel human site-specific DNA-binding factor has been partially purified from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA replication at the 3' end of the minute virus of mice (MVM) genome and functions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the origin sequence in a reaction which requires ATP hydrolysis. DNase I and copper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the entire region in the minimal left-end origin not already known to be occupied by NS1. Methylation and carboxy-ethylation interference analysis identified two ACGT motifs, spaced by five nucleotides, as the sequences responsible for this binding. A series of mutant oligonucleotides was then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demonstrate that the two motifs comprise a single binding site rather than two separate sites. Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cellular factor is responsible for both mobility shift and nicking activities. UV cross-linking and relative mobility assays suggest that PIF binds DNA as a heterodimer or higher-order multimer with subunits in the 80- to 100-kDa range.

Animals↗

PubMed2002

Lack of strand bias in UV-induced mutagenesis in Escherichia coli.

We have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in the lacZ gene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of the lacZ gene with respect to the origin of DNA replication. Depending on this orientation, any lacZ target sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis in uvrA strains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on the Escherichia coli chromosome may occur with equal or similar probability on leading and lagging strands.

DNA Replication↗