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PubMed2004

Protein and sequence requirements for the recruitment of the human origin recognition complex to the latent cycle origin of DNA replication of Epstein-Barr virus oriP.

Initiation of DNA replication from within the Epstein-Barr virus (EBV) latent cycle origin oriP occurs once per cell cycle and is almost entirely dependent upon cellular proteins. The human origin recognition complex (ORC) is recruited to oriP and orchestrates the events that lead to the initiation of replication. EBNA-1, the sole viral protein required for oriP-plasmid replication, binds four sites within the replicator but the role(s) it plays in the replication of oriP plasmids has not been elucidated. We investigated the recruitment of ORC to oriP in vivo and show that the binding of EBNA-1 to the replicator is necessary for the association of the ORC subunit Orc2 with the replicator. The minimal replicator of oriP consists of two EBNA-1 binding sites flanked by perfect 14-bp inverted repeats (a and b), but these repeats are dispensable for the association of Orc2 with the replicator. A mutational analysis of the 14-bp repeats provided additional support for a role for the telomere repeat binding protein 2 in oriP replicator function. We show that nucleotide differences between the oriP replicator of the B95-8 and Raji EBV genomes are not solely responsible for the inefficient utilization of this origin in the Raji EBV genome.

Binding Sites↗
PubMed1983

Binding of SV40 a protein to the BK virus origin of DNA replication.

The SV40 A protein (T antigen) binds to the putative origin of replication of the related BK virus (BKV). Protection studies with dimethyl sulfate identify multiple pentanucleotide contact sites in three distinct binding regions. In addition to the consensus family of recognition sequences, 5'-(G greater than T)(A greater than G)GGC-3', previously identified in the origin of SV40, BKV contains a contact 5'-AAGGC-3' site. As in the case of SV40, each binding region contains recognition sequences in different arrangements. The high affinity region I contains two sites arranged as direct repetitions covering a span of 17 base pairs (bp). In the intermediate affinity region II, four pentanucleotides are oriented as inverted repetitions with a span of 23 bp. Low affinity region III has a single contact site that can direct binding of the A protein. These different arrangements of DNA contact sites determine different patterns of protein binding and DNase protection in the three regions. The findings support a model of A protein binding to DNA previously proposed for the SV40 origin and establish a basis for future studies on regulatory phenomena at the BKV origin.

Antigens, Polyomavirus Transforming↗
PubMed1995

cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin.

Papillomavirus DNA replication requires the viral trans-acting factors E1 and E2 in addition to the host cell's general replication machinery. The origins of DNA replication in bovine and human papillomavirus genomes have been localized to a specific part of the upstream regulatory region (URR) which includes recognition sites for E1 and E2 proteins. To fine map cis-acting elements influencing human papillomavirus type 11 (HPV-11) DNA replication and to determine the relative contributions of such sites, we engineered consecutive linker substitution mutations across a region of 158 bp in the HPV-11 origin and tested mutant origins for replication function in a cell-based transient replication assay. Our results both confirm and extend the findings of others. E2 binding sites are the major cis components of HPV-11 DNA replication, and there is evidence for synergy between these sites. Differential capacity of the three E2 binding sites within the origin to affect replication may be attributed, at least in part, to context. At least one E2 binding site is essential for replication. The imperfect AT-rich palindrome of the E1 helicase binding site is not essential since replication occurs even in the absence of this sequence. However, replication is enhanced by the presence of the palindromic sequence in the HPV-11 origin. Sequence components adjacent to the E1 and E2 binding sites, comprising AT-rich and purine-rich elements and the consensus TATA box sequence, probably contribute to the overall efficiency of replication, though they are nonessential. None of the other cis elements of the HPV-11 origin region analyzed seems to influence replication significantly in the system described. The HPV-11 origin of DNA replication therefore differs from those of the other papovaviruses, simian virus 40 and polyomavirus, inasmuch as an intact helicase binding site and adjacent AT-rich components, while influential, are not absolutely essential.

Animals↗
PubMed1993

Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29.

DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal region of approximately 400 bp which lies upstream of the gene that is homologous to herpes simplex virus UL29, a region that carries an origin in other betaherpesviruses and in some alphaherpesviruses.

DNA Mutational Analysis↗
PubMed1984

Unusual regulation of simian virus 40 early-region transcription in genomes containing two origins of DNA replication.

As part of our efforts to create multifunctional vectors for the transduction of animal cells, a set of simian virus 40 recombinants were constructed which contain an inverted duplication of the region including the origin of viral DNA replication (ori) and the early-region promoter. The unusual aspects of the structure of these recombinant genomes revealed several unexpected features of their function. In particular, transcription from the early-region promoters on these recombinants occurred primarily after the start of DNA replication, and, in that sense, these promoters behaved as if they were late-region promoters. This behavior results from the fact that these genomes contain multiple ori segments, and, therefore, they replicate earlier and faster than wild-type virus DNA, thereby causing a precocious shift in the initiation of early-region transcription from sites downstream of ori to sites located upstream of ori. The abnormal expression from multiple ori genomes is consistent with our present notions regarding the replication-dependent shift in early-region transcriptional start sites (Buchman et al., Mol. Cell. Biol. 4:1900-1914). Since our experiments demonstrate that RNAs initiated upstream of ori contribute to T-antigen formation late in infection, we suggest that the shift in early-region transcription starts modulates large T-antigen production in concert with viral DNA replication.

Animals↗
PubMed1995

Molecular cloning and complete nucleotide sequence of the repeated unit and flanking gene of the scallop Pecten maximus mitochondrial DNA: putative replication origin features.

In the bivalve mollusc Pecten maximus, the size of the mitochondrial DNA molecules ranges from 20 to 25.8 kbp. This variability is mainly correlated with the occurrence of a variable domain composed with two to five 1.6-kbp repeated units tandemly arrayed in the genome. DNA fragments spanning the 1,586-base-pair-long repeated element and the nearest flanking gene have been cloned and sequenced. This sequence was analyzed regarding its base composition and potential secondary structures. The repeated unit domain was positioned and oriented with regard to the known flanking gene. It ends 2 base pairs upstream relative to the beginning of the tRNAgly gene. The peculiar properties of the repeated unit were compared with those of the 1,442-bp repeated element found in the mitochondrial genome of the deep sea scallop Placopecten magellanicus. This comparison provided evidence for the absence of nucleotide conservation, except for a small sequence engaged in a secondary structure, but argued for a strong pressure maintaining domains with specific nucleotide content. A possible role for the conserved sequence is discussed.

Animals↗
PubMed2005

Origins of DNA replication in the three domains of life.

Replication of DNA is essential for the propagation of life. It is somewhat surprising then that, despite the vital nature of this process, cellular organisms show a great deal of variety in the mechanisms that they employ to ensure appropriate genome duplication. This diversity is manifested along classical evolutionary lines, with distinct combinations of replicon architecture and replication proteins being found in the three domains of life: the Bacteria, the Eukarya and the Archaea. Furthermore, although there are mechanistic parallels, even within a given domain of life, the way origins of replication are defined shows remarkable variation.

Archaea↗
PubMed2003

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins.

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.

Base Sequence↗
PubMedDate not supplied

Bent DNA at a yeast autonomously replicating sequence.

DNA fragments that show retarded electrophoretic mobility through polyacrylamide gels have been found in both prokaryotes and eukaryotes. In the case of kinetoplast DNA, evidence has been presented that the DNA is curved or 'bent'. Bent DNA has previously been found at the lambda and simian virus 40 (SV40) DNA replication origins. Here we show the existence of bent DNA at a yeast autonomously replicating sequence (ARS1), a putative replication origin. The bent DNA has been localized to a 40-55 base pair (bp) segment and contains six (A)3-5 stretches (that is, six poly(A) stretches, three to five nucleotides in length) phased approximately every 10.5 bp. This region contains a DNA binding site for a yeast protein factor. This site lies at the 3' end of the TRP1 gene, in a region devoid of nucleosomes, and is positioned 80 bp away from the ARS consensus sequence; removal of this region impairs ARS function in vivo. The bent DNA may be involved in transcription termination or the prevention of nucleosome assembly in this region.

Base Sequence↗
PubMed1999

Universal minicircle sequence-binding protein, a sequence-specific DNA-binding protein that recognizes the two replication origins of the kinetoplast DNA minicircle.

Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatid Crithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously described universal minicircle sequence (UMS)-binding protein (UMSBP) (Tzfati, Y., Abeliovich, H., Avrahami, D., and Shlomai, J. (1995) J. Biol. Chem. 270, 21339-21345). This CCHC-type zinc finger protein binds the single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification from C. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes. Stoichiometry analysis indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.

Animals↗
PubMed2006

Demonstration of nicking/joining activity at the origin of DNA replication associated with the rep and rep' proteins of porcine circovirus type 1.

The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3'-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a double-stranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep' have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep' is as yet unknown. In this study, the ability of PCV1 Rep and Rep' to "nick" and "join" strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5'-TAGTATTAC-3') located at the apex of a putative stem-loop structure. In addition, the Rep and Rep' proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual "nicking/joining" activities associated with PCV1 Rep and Rep' are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.

Base Pairing↗
PubMed1993

Identification of EBNA1 amino acid sequences required for the interaction of the functional elements of the Epstein-Barr virus latent origin of DNA replication.

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin, oriP. This activation involves the direct interaction of EBNA1 dimers with multiple sites within the two noncontiguous functional elements of the origin, the family of repeats (FR) element and the dyad symmetry (DS) element. The efficient interaction of EBNA1 dimers bound to these two elements in oriP results in the formation of DNA loops in which the FR and DS elements are bound together through EBNA1. In order to elucidate the mechanism by which EBNA1 induces oriP DNA looping, we have investigated the DNA sequences and EBNA1 amino acids required for EBNA1-mediated DNA looping. Using a series of truncation mutants of EBNA1 produced in baculovirus and purified to apparent homogeneity, we have demonstrated that the EBNA1 DNA binding and dimerization domain is not sufficient to mediate oriP DNA looping and that an additional region(s) located between amino acids 346 and 450 is required. Single EBNA1-binding sites, separated by 930 bp of plasmid DNA, were also shown to support EBNA1-mediated looping, indicating that the formation of large EBNA1 complexes, such as those observed on oriP FR and DS elements, is not a requirement for looping.

Amino Acid Sequence↗
PubMed2005

The MCM complex: its role in DNA replication and implications for cancer therapy.

The MCM complex controls the once per cell cycle DNA replication in eukaryotic cells. In a process known as DNA replication licensing, it primes chromatin for DNA replication by binding origins of DNA replication during the late M to early G1 phase of the cell cycle. Activated by S phase promoting protein kinases, the origin-bound MCM complexes unwind the double stranded DNA at the origins, recruit DNA polymerases and initiate DNA synthesis. Coupled with the initiation of DNA replication in the S phase, the MCM complexes move away from replication origins as a component of the DNA replication fork, likely serving as DNA helicases. Their departure deprives replication origins the ability to re-initiate DNA replication for the reminder of the cell cycle. Because of its vital role in genome duplication in proliferating cells, deregulation of the MCM function results in chromosomal defects that may contribute to tumorigenesis. The MCM proteins are highly expressed in malignant human cancers cells and pre-cancerous cells undergoing malignant transformation. They are not expressed in differentiated somatic cells that have been withdrawn from the cell cycle. Therefore, these proteins are ideal diagnostic markers for cancer and promising targets for anti-cancer drug development. In this article, I will overview the structures and functions of the MCM complex with an effort to integrate insights from recent biochemical and structural studies. Discussions will also cover activities and structures of the complex that may be useful for the development of drug screens.

Animals↗
PubMed2002

Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication.

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.

Animals↗
PubMed1991

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.

Animals↗
PubMed1994

Stabilization of the EBNA1 protein on the Epstein-Barr virus latent origin of DNA replication by a DNA looping mechanism.

DNA replication from the Epstein-Barr virus latent origin of replication, oriP, is activated by Epstein-Barr nuclear antigen 1 (EBNA1). This activation involves the binding of EBNA1 dimers to multiple sites present in the two noncontiguous functional elements of oriP, the dyad symmetry element (DS) from which replication initiates, and the family of repeats (FR) enhancer element. EBNA1 complexes formed on the FR and DS elements of oriP interact by a DNA looping mechanism. This interaction requires EBNA1 sequences in addition to those required for DNA binding and dimerization. To map the EBNA1 sequences required for the efficient interaction of FR- and DS-bound EBNA1 complexes, we have overproduced in Escherichia coli and purified a series of EBNA1 N-terminal truncation mutants, all of which retain the DNA binding and dimerization domains. The results of electron microscopy and ligation-enhancement assays using these mutants indicated that EBNA1 sequences between amino acids 350 and 361 are required for the efficient interaction of FR- and DS-bound EBNA1 complexes. EBNA1-mediated FR-DS interactions were shown to stabilize EBNA1 binding to the DS element, while EBNA1-mediated DS-DS interactions did not. These results suggest that the stabilization of EBNA1 on the DS element, which occurs as a result of EBNA1-mediate oriP looping, may be important for the activation of DNA replication from the DS element.

Antigens, Viral↗
PubMedDate not supplied

Mouse p53 inhibits SV40 origin-dependent DNA replication.

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.

Animals↗