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At least 973 records · Page 54Linked to original sources
PubMed1986

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.

Animals↗
PubMed2006

Spatial and temporal organization of the Bacillus subtilis replication cycle.

DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.

Bacillus subtilis↗
PubMed1982

Conserved sequences at the origin of adenovirus DNA replication.

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A . T-rich region which is partially conserved among these serotypes, and a distal G . C-rich region which is less well conserved. The G . C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis.

Adenoviruses, Human↗
PubMed1992

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus.

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

Bacterial Proteins↗
PubMed2003

Analysis of the intrinsic bend in the M13 origin of replication by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40-50 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 +/- 8 and 62 +/- 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.

Crystallography↗
PubMed1992

Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication.

Chinese hamster genomic DNA sequences from the region downstream of the dihydrofolate reductase (DHFR) gene reported to contain a chromosomal origin of bidirectional DNA replication (OBR-1) were tested for their ability to support autonomous DNA replication in human cells. A 13.3 kilobase fragment containing OBR-1 and surrounding sequences supported replication in short-term and long-term replication assays, while a 4.5 kb fragment containing OBR-1 did not support substantial replication in either assay. These results are consistent with our previous observations that large fragments of human DNA support replication, while smaller fragments are less efficient. The replication activities of plasmids containing OBR-1 were no greater than those of randomly chosen human fragments of similar size. Furthermore, two-dimensional gel analysis of plasmids containing OBR-1 indicated that initiation does not preferentially occur within the OBR-1 region. These results suggest that in the context of autonomous replication, the DHFR sequences tested do not contain genetic information specifying site-specific replication initiation. Possible implications of these results for chromosomal replication are discussed.

Animals↗
PubMed2006

rmlB and rmlC genes are essential for growth of mycobacteria.

The rhamnose-GlcNAc disaccharide linker is fundamental to the structural integrity of mycobacterial cell wall. The donor dTDP-rhamnose is synthesized by four enzymes (RmlA, B, C, and D) beginning with dTTP and glucose-1-phosphate. We generated M. smegmatis rmlB gene knockout mutant (transcription of downstream rmlC gene was blocked because of a polar effect) by homologous recombination. When the Mycobacterium tuberculosis (Tb) rmlB rescue plasmid carrying a temperature-sensitive replication origin and Tb rmlC bearing plasmid with a normal replication origin were present in the mc(2)155 rmlB knockout mutant, the mutant was unable to grow at non-permissive temperature (42 degrees C) where the Tb rmlB rescue plasmid is lost. While the Tb rmlC rescue plasmid carrying a temperature-sensitive replication origin and Tb rmlB bearing plasmid with a normal replication origin were present in the mc(2)155 rmlB knock out mutant, this mutant was also unable to grow at the non-permissive temperature where the Tb rmlC rescue plasmid is lost. These results demonstrate that rmlB and rmlC genes are essential for mycobacterial growth, therefore, RmlB and RmlC are essential targets to develop new anti-tuberculosis drugs.

Cell Proliferation↗
PubMed1996

The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.

Animals↗
PubMed1998

Unwinding of the Escherichia coli origin of replication (oriC) can occur in the absence of initiation proteins but is stabilized by DnaA and histone-like proteins IHF or HU.

The unwinding of the origin of replication (oriC) is a critical step in initiation of DNA replication in Escherichia coli. Previous observations indicate that efficient unwinding of supercoiled plasmid templates containing oriC sequences requires the DnaA initiation protein and one or more accessory factors. The precise contribution of each protein to this process is unknown. Here, we demonstrate that unwinding can occur under physiological conditions at the same bases in oriC, in either the presence or the absence of initiation proteins, as detected by a single-strand specific nuclease, P1. This suggests that oriC unwinding is a spontaneous event determined solely by DNA sequence. DnaA and IHF, as part of a large nucleoprotein complex, may function to stabilize the DNA strand opening prior to initiation of DNA replication.

Adenine↗
PubMed1991

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.

Bacterial Proteins↗
PubMed1992

The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.

Base Sequence↗
PubMed1988

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.

Animals↗
PubMed1988

Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.

Replication of simian virus 40 (SV40) DNA is dependent upon the binding of the viral T-antigen to the SV40 origin of replication. Structural changes in the origin of replication induced by binding of T-antigen were probed by chemical modifications of the DNA. In the presence of ATP, T-antigen rendered two of three domains in the SV40 core origin hypersensitive to attack by either dimethyl sulfate or potassium permanganate (KMnO4). One of these domains, the early palindrome, was shown to contain an 8-bp region of melted DNA as determined from methylation of cytosine residues and by nuclease S1 cleavage of methylated DNA. DNA melting was not dependent upon either the hydrolysis of ATP or the binding of T-antigen to an adjacent site (site I). A second domain, the A/T element, was extensively modified by KMnO4 but no significant melting was detected. Rather, the pattern of modification indicates that T-antigen caused a conformational change of the double-stranded DNA in this region. These results suggest that T-antigen, in the presence of ATP, destabilizes the SV40 origin by melting and structurally deforming two flanking regions within the core origin sequence. These DNA structural changes may provide access to other replication factors, allowing complete denaturation of the SV40 origin and the initiation of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed2001

The replicator of the Epstein-Barr virus latent cycle origin of DNA replication, oriP, is composed of multiple functional elements.

Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as oriP. The replicator of oriP contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of oriP plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for an oriP plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator of oriP consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.

Base Sequence↗
PubMed2006

Structural basis for the cooperative assembly of large T antigen on the origin of replication.

Large T antigen (LTag) from simian virus 40 (SV40) is an ATP-driven DNA helicase that specifically recognizes the core of the viral origin of replication (ori), where it oligomerizes as a double hexamer. During this process, binding of the first hexamer stimulates the assembly of a second one. Using electron microscopy, we show that the N-terminal part of LTag that includes the origin-binding domain does not present a stable quaternary structure in single hexamers. This disordered region, however, is well arranged within the LTag double hexamer after specific ori recognition, where it mediates the interactions between hexamers and constructs a separated structural module at their junction. We conclude that full assembly of LTag hexamers occurs only within the dodecamer, and requires the specific hexamer-hexamer interactions established upon binding to the origin of replication. This mechanism provides the structural basis for the cooperative assembly of LTag double hexamer on the cognate viral ori.

Antigens, Polyomavirus Transforming↗
PubMed1999

The 25 kDa protein recognizing the rat curved region upstream of the origin of the L-strand replication is the rat homologue of the human mitochondrial transcription factor A.

Mass spectrometry matrix-assisted laser desorption ionization (MALDI) analysis and N-terminus sequencing as well as immunoblotting experiments using human and mouse antibodies have allowed us to identify the 25 kDa protein, previously isolated from rat liver using magnetic beads coated with a rat liver mitochondrial (mt) DNA region upstream of the Ori-L, as the homologue of human mt transcription factor A (mtTFA). We can therefore identify this DNA binding protein as the rat mtTFA. Furthermore, since we previously showed that the 25 kDa protein purified from rat liver was able to bind the curved mtDNA region upstream of the Ori-L as well as the curved mtDNA in the D-loop region, the results here reported lead us to state, for the first time, that mtTFA binds both the curved regions of mtDNA upstream of the two replication origins.

Amino Acid Sequence↗
PubMed1999

HMR-I is an origin of replication and a silencer in Saccharomyces cerevisiae.

There appear to be fundamental differences between the properties of the silencers at HML and HMR, with some being origins of replication and others not. Moreover, past studies have suggested that HMR-I's role in silencing may be restricted to plasmid contexts. This study established that HMR-I, like HMR-E and unlike either HML silencer, is an origin of replication. Moreover, both HMR-E and HMR-I contribute to silencing of a chromosomal HMR locus. In addition, we found that Abf1p plays no unique role in silencer function.

DNA Replication↗
PubMed2003

Interaction of EBV latent origin of replication with the nuclear matrix: identification of S/MAR sequences and protein components.

During latency, Epstein Barr virus (EBV) genome, as an episome, is attached to the nuclear matrix (NM) via the latent origin of replication ori P. Within this element, we have found that a region, 580 bp long, encompassing the replicator DS element, shows the strongest affinity for the NM. In addition, by cross-linking with cis-diamminedichloroplatinum, we have identified two NM proteins with an apparent molecular weight of 85 and 60 kDa that, with high affinity and specificity, bind ori P. These proteins are not induced by EBV infection, but their interaction with ori P is lost upon induction of EBV lytic cycle. These data strongly suggest that the binding of ori P to specific components of the NM is required for EBV latent replication.

Binding Sites↗