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PubMed1984

Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a messenger RNA and a transfer RNA.

The initiation of transcription of the yeast mitochondrial genes coding for subunit I of cytochrome c oxidase (COX1) and for tRNA1Thr has been examined. COX1 messenger RNA synthesis is initiated in a conserved nonanucleotide sequence (ATATAAGTA) which we have previously found immediately upstream of ribosomal RNA genes at positions at which RNA synthesis starts. The 5'-end of the precursor of tRNA1Thr is located in a variant nonanucleotide motif (TTATAAGTA), which may be characteristic for tRNA genes. Using a partially purified fraction of mtRNA polymerase, we demonstrate that RNA synthesis is precisely initiated in vitro in nonanucleotide sequences preceding both ribosomal RNA-, tRNA- and messenger RNA-encoding genes and origins of replication.

Base Sequence↗

PubMed1988

Bent DNA structures associated with several origins of replication are recognized by a unique enzyme from trypanosomatids.

Sequence-directed bending of the DNA double helix is a conformational variation found in both prokaryotic and eukaryotic organisms. The utilization of bent DNA structures from various sources as specific signals recognized by an enzyme is demonstrated here using a unique endonuclease purified from trypanosomatid cells. Crithidia fasciculata nicking enzyme was previously shown to recognize specifically the bent structure found in kinetoplast DNA minicircles. The binding constant measured for this specific interaction is of two orders of magnitude higher than that measured for the binding of the enzyme to a non-curved sequence. As determined by binding competition and mobility shift electrophoresis analyses, this enzyme recognizes the sequence-directed bends associated with the origins of replication of bacteriophage lambda and simian virus 40 (SV40), as well as that located within the autonomously replicating sequence (ARS1) region of the yeast S. cerevisiae.

Animals↗

PubMed1984

Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA.

Polyomavirus large T antigen binds specifically to multiple sites within the regulatory region of the viral genome. Experiments done with crude extracts from wild-type virus-infected mouse cells and immunoprecipitation of protein-DNA complexes localized two high-affinity binding sites on the early region side of the DNA replication origin. Purification of the large T antigen by immunoaffinity chromatography made it possible to refine the analysis through application of DNase I footprinting. The high-affinity interactions were resolved into three closely spaced, but distinct, binding regions. These begin at a site only slightly overlapping the early boundary of the core replication origin, a location highly homologous to that of simian virus 40 large T antigen-binding site I, but then extend away from the origin toward the early coding sequence and thus span the early region transcriptional initiation sites. Each tight-binding region contains from two to four copies of the sequence 5'-(A = T)G(A greater than G)GGC-3' repeated at 9- to 11-base-pair spacing. At high protein concentrations and at low ionic strength, additional sites within the core replication origin and in the enhancer region were protected from DNase I digestion. These minor binding sites also included repeats of sequences related to the consensus, but at different spacings. Our results suggest that, unlike simian virus 40 DNA, the polyomavirus genome may have distinct regions of interaction with its large T antigen which separately are involved in initiation of DNA replication and the regulation of viral transcription.

Antigens, Viral, Tumor↗

PubMed1993

Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444.

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5 alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C. perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C. acetobutylicum and C. perfringens suggested that it was identical to that present in E. coli transformants.

Bacteriophages↗

PubMed2002

Two mcm3 mutations affect different steps in the initiation of DNA replication.

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.

Alleles↗

PubMed1992

The virulence-determining genomic BamHI fragment 4 of pseudorabies virus contains genes corresponding to the UL15 (partial), UL18, UL19, UL20, and UL21 genes of herpes simplex virus and a putative origin of replication.

The genomic BamHI-fragment 4 of pseudorabies virus (PrV) has previously been shown to encode functions necessary for expression of PrV neurovirulence (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1984, J. Virol. 52, 198-205). To identify proteins that might be involved in the neurotropism of PrV we sequenced the complete 9382-bp fragment BamHI-4, the longest contiguous sequence determined in the UL region of PrV so far, and analyzed its coding capacity. In an arrangement similar to that found in herpes simplex virus type 1 we identified complete open reading frames encoding proteins with strong homology to the UL18 (50% homology), UL19 (60% homology), UL20 (33% homology), and UL21 (36% homology) polypeptides and the 3'-part of a gene homologous to UL15 (67% homology) of HSV-1. In addition, a consensus sequence for an alphaherpesviral origin of replication was found at the left terminus of the fragment.

Base Sequence↗

PubMed1988

Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication.

The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC, and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity (S. Deb, A.L. DeLucia, C.-P. Baur, A. Koff, and P. Tegtmeyer, Mol. Cell. Biol. 6:1663-1670, 1986), but its functional role was not defined. In the present report, we have used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlated with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.

Animals↗

PubMed1989

Locations of nucleosomes on the regulatory region of simian virus 40 chromatin.

We have asked where the nucleosomes are located with respect to the replication origin and regulatory region of simian virus 40 DNA, what would be the possible functional consequences of the identified locations, and to what extent these locations correlate with the current views on mechanisms involved in establishing nucleosome-free regions in chromatin. To identify the precise location of nucleosomes, we have shot-gun cloned and sequenced nucleosomal DNA obtained from micrococcal nuclease digestion of wt776 chromatin prepared late in infection. Our results indicate that nucleosomes do not occupy unique positions over the replication origin or the elements involved in transcriptional control. However, it appears that the nucleosome distribution is not random, since several nucleosomes are represented by two or more independently generated clones. Two nearly identical cloned fragments map over the replication origin; five include 1.5 copies of the 72 base-pair enhancer sequences; and eight map to a region that spans a DNA bending locus and the major transcription initiation site of the late genes. The complex nucleosome distribution pattern observed in our direct analysis suggests that disparate nucleosome-free regions may be involved in controlling replication, and selective expression of the viral early or late genes.

Cell Line↗

PubMed1978

Human mitochondrial DNA: analysis of 7S DNA from the origin of replication.

Heat-treated samples of human mitochondrial DNA (mtDNA) exhibited a set of three low molecular weight DNA bands in addition to the major mtDNA band when electrophoresed in polyacrylamide gels. These DNA components were seen only after heat treatment or after relaxation of the mtDNA with a restriction endonuclease. The three components were single stranded and had sizes of 550, 585, and 629 nucleotides, close to the size (600 nucleotides) estimated from contour length measurements for the 7S DNA from the D loop of human mtDNA. Hybridization of the components with restriction endonuclease fragments of known position in the mtDNA confirmed this identification. Digestion of each 7S DNA component with the restriction endonuclease Hae III produced three fragments, two of which were identical in size among the components and the third of which varied. This third fragment, shown to be from the 5' end of each component, differed in length by approximately 35 nucleotides among the components. These results suggest that human 7S mtDNA synthesis is terminated at a distinct position and that it is either initiated at one of three possible sites in the same mtDNA or that the mtDNA population consists of three subpopulations, each differing from the others by the presence or absence of a nucleotide sequence immediately adjacent to the origin of replication.

DNA Restriction Enzymes↗

PubMed2005

Nonrandom clusters of palindromes in herpesvirus genomes.

Palindromes are symmetrical words of DNA in the sense that they read exactly the same as their reverse complementary sequences. Representing the occurrences of palindromes in a DNA molecule as points on the unit interval, the scan statistics can be used to identify regions of unusually high concentration of palindromes. These regions have been associated with the replication origins on a few herpesviruses in previous studies. However, the use of scan statistics requires the assumption that the points representing the palindromes are independently and uniformly distributed on the unit interval. In this paper, we provide a mathematical basis for this assumption by showing that in randomly generated DNA sequences, the occurrences of palindromes can be approximated by a Poisson process. An easily computable upper bound on the Wasserstein distance between the palindrome process and the Poisson process is obtained. This bound is then used as a guide to choose an optimal palindrome length in the analysis of a collection of 16 herpesvirus genomes. Regions harboring significant palindrome clusters are identified and compared to known locations of replication origins. This analysis brings out a few interesting extensions of the scan statistics that can help formulate an algorithm for more accurate prediction of replication origins.

Data Interpretation, Statistical↗

PubMed1995

Murine polyomavirus and simian virus 40 large T antigens produce different structural alterations in viral origin DNA.

Murine polyomavirus (Py) and simian virus (SV40) encode homologous large T antigens (T Ags) and also have comparable sequence motifs in their core replication origins. While the ability of SV40 T Ag to produce specific distortions within the SV40 core replication origin (ori) in a nucleotide-dependent fashion has been well documented, little is known about related effects of Py T Ag on Py ori DNA. Therefore, we have examined viral origin DNA binding in the presence of nucleotide and the resulting structural changes induced by Py and SV40 T Ags by DNase I footprinting and KMnO4 modification assays. The structural changes in the Py ori induced by Py T Ag included sites within both the A/T and early side of the core origin region, consistent with what has been shown for SV40. Interestingly, however, Py T Ag also produced sites of distortion within the center of the origin palindrome and at several sites within both the early and late regions that flank the core ori. Thus, Py T Ag produces a more extensive and substantially different pattern of KMnO4 modification sites than does SV40 T Ag. We also observed that both T Ags incompletely protected and distorted the reciprocal ori region. Therefore, significant differences in the interactions of Py and SV40 T Ags with ori DNA may account for the failure of each T Ag to support replication of the reciprocal ori DNA in permissive cell extracts.

Animals↗

PubMed1999

The chromatin structure of the long control region of human papillomavirus type 16 represses viral oncoprotein expression.

The long control region (LCR) of human papillomavirus type 16 (HPV-16) has a size of 850 bp (about 12% of the viral genome) and regulates transcription and replication of the viral DNA. The 5' segment of the LCR contains transcription termination signals and a nuclear matrix attachment region, the central segment contains an epithelial cell-specific enhancer, and the 3' segment contains the replication origin and the E6 promoter. Here we report observations on the chromatin organization of this part of the HPV-16 genome. Treatment of the nuclei of CaSki cells, a cell line with 500 intrachromosomal copies of HPV-16, with methidiumpropyl-EDTA-Fe(II) reveals nucleosomes in specific positions on the LCR and the E6 and E7 genes. One of these nucleosomes, which we termed Ne, overlaps with the center of the viral enhancer, while a second nucleosome, Np16, overlaps with the replication origin and the E6 promoter. The two nucleosomes become positioned on exactly the same segments after in vitro assembly of chromatin on the cloned HPV-16 LCR. Primer extension mapping of DNase I-cleaved chromatin revealed Np16 to be positioned centrally over E6 promoter elements, extending into the replication origin. Ne covers the center of the enhancer but leaves an AP-1 site, one of the strongest cis-responsive elements of the enhancer, unprotected. Np16, or a combination of Np16 and Ne, represses the activity of the E6 promoter during in vitro transcription of HPV-16 chromatin. Repression is relieved by addition of Sp1 and AP-1 transcription factors. Sp1 alters the structure of Np16 in vitro, while no changes can be observed during the binding of AP-1. HPV-18, which has a similar arrangement of cis-responsive elements despite its evolutionary divergence from HPV-16, shows specific assembly in vitro of a nucleosome, Np18, over the E1 binding site and E6 promoter elements but positioned about 90 bp 5' of the position of Np16 on the homologous HPV-16 sequences. The chromatin organization of the HPV-16 and HPV-18 genomes suggests important regulatory roles of nucleosomes during the viral life cycle.

Chromatin↗

PubMed1997

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.

Animals↗

PubMed1997

Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids.

Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids. Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094. The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced. Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences. Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA. In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA. A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication. This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

Amino Acid Sequence↗

PubMed1995

Characterization and replication mode determination of the minimal replicon of Tetragenococcus halophila ATCC33315 plasmid pUCL287.

pUCL287 is a cryptic plasmid of Tetragenococcus halophila (formerly Pediococcus halophilus) ATCC33315 of relatively small size (8.7 kb). Its minimal replicon was located on a 1235 bp MamI-EcoRI fragment. This minimal replicon contains a non-translated region, followed by a gene encoding a putative 311 amino acid protein. Deletion experiments showed that the non-translated region corresponds to the replication origin. Determination of the replication mode was carried out in Enterococcus faecalis JH2-2 harboring pUCL287 minimal replicon. The replicating intermediates detected revealed that pUCL287 minimal replicon follows a bidirectional theta replicating mode.

Amino Acid Sequence↗

PubMed2005

Preventing re-replication of chromosomal DNA.

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.

Animals↗

PubMed2002

Escherichia coli DnaA protein loads a single DnaB helicase at a DnaA box hairpin.

The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure.

Bacterial Proteins↗

PubMed2004

Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.

Binding Sites↗