Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Origin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed1999

Genetic methods for characterizing the cis-acting components of yeast DNA replication origins.

Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species. Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function. Although alternative methods are available for genetic characterization of replication origins in other organisms, the simplicity of the autonomous replication assay in yeasts has permitted development of the deepest understanding to date of eukaryotic replication origin structure. This information has come primarily from studies with Saccharomyces cerevisiae. However, there are many other yeast species, each with its own variety of replication origins. Use of the methods summarized here to characterize origins in other yeast species is likely to provide additional insights into eukaryotic replication origin structure.

Cell Division↗
PubMed1998

Multiple cis elements contribute to geminivirus origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied in trans. These experiments demonstrated that the minimal origin is located in 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additional cis elements--one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replication in vivo and AL1/DNA binding in vitro. Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multiple cis elements that are likely to interact with each other during initiation of replication.

Base Sequence↗
PubMed1989

Estimates of the maximum time required to originate life.

Fossils of the oldest microorganisms exist in 3.5 billion year old rocks and there is indirect evidence that life may have existed 3.8 billion years ago (3.8 Ga). Impacts able to destroy life or interrupt prebiotic chemistry may have occurred after 3.5 Ga. If large impactors vaporized the oceans, sterilized the planets, and interfered with the origination of life, life must have originated in the time interval between these impacts which increased with geologic time. Therefore, the maximum time required for the origination of life is the time that occurred between sterilizing impacts just before 3.8 Ga or 3.5 Ga, depending upon when life first appeared on Earth. If life first originated 3.5 Ga, and impacts with kinetic energies between 2 x 10(34) and 2 x 10(35) were able to vaporize the oceans, using the most probable impact flux, we find that the maximum time required to originate life would have been 67 to 133 million years (My). If life first originated 3.8 Ga, the maximum time to originate life was 2.5 to 11 My. Using a more conservative estimate for the flux of impacting objects before 3.8 Ga, we find a maximum time of 25 My for the same range of impactor kinetic energies. The impact model suggest that it is possible that life may have originated more than once.

Earth, Planet↗
PubMed2006

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid.

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.

Bacterial Proteins↗
PubMed1998

CLB5-dependent activation of late replication origins in S. cerevisiae.

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Chromosomes, Fungal↗
PubMed1999

DNA methylation at mammalian replication origins.

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.

Animals↗
PubMed2004

Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.

A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close ( approximately 10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase.

Animals↗
PubMed2005

Bacteriophage T4 helicase loader protein gp59 functions as gatekeeper in origin-dependent replication in vivo.

Bacteriophage T4 initiates origin-dependent replication via an R-loop mechanism in vivo. During in vitro reactions, the phage-encoded gp59 stimulates loading of the replicative helicase, gp41, onto branched intermediates, including origin R-loops. However, although gp59 is essential for recombination-dependent replication from D-loops, it does not appear to be required for origin-dependent replication in vivo. In this study, we have analyzed the origin-replicative intermediates formed during infections that are deficient in gp59 and other phage replication proteins. During infections lacking gp59, the initial replication forks from two different T4 origins actively replicated both leading- and lagging-strands. However, the retrograde replication forks from both origins were abnormal in the gp59-deficient infections. The lagging-strand from the initial fork was elongated as a new leading-strand in the retrograde direction without lagging-strand synthesis, whereas in the wild-type, leading- and lagging-strand synthesis appeared to be coupled. These results imply that gp59 inhibits the polymerase holoenzyme in vivo until the helicase-primase (gp41-gp61) complex is loaded, and we thereby refer to gp59 as a gatekeeper. We also found that all origin-replicative intermediates were absent in infections deficient in the helicase gp41 or the single-strand-binding protein gp32, regardless of whether gp59 was present or absent. These results argue that replication from the origin in vivo is dependent on both the helicase and single-strand-binding protein and demonstrate that the strong replication defect of gene 41 and 32 single mutants is not caused by gp59 inhibition of the polymerase.

Bacteriophage T4↗
PubMed2001

The positioning and dynamics of origins of replication in the budding yeast nucleus.

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

Cell Cycle↗
PubMed1994

Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe.

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.

Chromosome Mapping↗
PubMed2000

Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding.

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G(1). To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.

Adenosine Triphosphatases↗
PubMed1999

Activation of dormant origins of DNA replication in budding yeast.

Eukaryotic genomes often contain more potential replication origins than are actually used during S phase. The molecular mechanisms that prevent some origins from firing are unknown. Here we show that dormant replication origins on the left arm of budding yeast chromosome III become activated when both passive replication through them is prevented and the Mec1/Rad53 checkpoint that blocks late-origin firing is inactivated. Under these conditions, dormant origins fire very late relative to other active origins. These experiments show that some dormant replication origins are competent to fire during S phase and that passage of a replication fork through such origins can inactivate them.

Cell Cycle↗
PubMed1999

Two new early bacteriophage T4 genes, repEA and repEB, that are important for DNA replication initiated from origin E.

Two new, small, early bacteriophage T4 genes, repEA and repEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:83-98, 1995). Therefore, repEA and repEB were expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two repE genes, we constructed nonsense mutations in each of them and combined them with the motA mutation sip1 that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the single motA mutant, but early transcription of oriE was reduced neither in the motA nor in the repE mutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for repEA and repEB. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons.

Amino Acid Sequence↗
PubMed2003

Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin.

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.

Adenoviridae↗
PubMed1997

Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.

Base Sequence↗
PubMed2006

Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa.

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.

Binding Sites↗
PubMed2005

Identification of replication origins in archaeal genomes based on the Z-curve method.

The Z-curve is a three-dimensional curve that constitutes a unique representation of a DNA sequence, i.e., both the Z-curve and the given DNA sequence can be uniquely reconstructed from the other. We employed Z-curve analysis to identify one replication origin in the Methanocaldococcus jannaschii genome, two replication origins in the Halobacterium species NRC-1 genome and one replication origin in the Methanosarcina mazei genome. One of the predicted replication origins of Halobacterium species NRC-1 is the same as a replication origin later identified by in vivo experiments. The Z-curve analysis of the Sulfolobus solfataricus P2 genome suggested the existence of three replication origins, which is also consistent with later experimental results. This review aims to summarize applications of the Z-curve in identifying replication origins of archaeal genomes, and to provide clues about the locations of as yet unidentified replication origins of the Aeropyrum pernix K1, Methanococcus maripaludis S2, Picrophilus torridus DSM 9790 and Pyrobaculum aerophilum str. IM2 genomes.

Archaea↗
PubMed1998

Regulation of mammalian replication origin usage in Xenopus egg extract.

Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

Animals↗