Both the conserved stem-loop and divergent 5'-flanking sequences are required for initiation at the human mitochondrial origin of light-strand DNA replication

Mammalian mitochondrial DNAs contain a conserved origin of light-strand replication that supports accurate initiation of DNA synthesis in vitro. This provides an opportunity to examine the sequence requirements for initiation through in vitro analysis of a series of deleted and mutagenized DNA templates. These assays use enzymes isolated from human mitochondria and single-stranded DNA templates containing deletions or substitutions in the known origin region. The data indicate that accurate and efficient light-strand replication in vitro requires the previously identified stem-loop structure located within a tRNA cluster. In addition, the template sequence 3'-GGCCG-5', located immediately adjacent to the stem, is necessary for efficient replication. This sequence, the complement of which encodes the 3' end of tRNACys, may be the site of transition from RNA primer synthesis to DNA synthesis. Surprisingly, substitutions within a region located in the loop of this origin do not reduce levels of replication.     

Mechanism of mitochondrial DNA replication in mouse L-cells: localization and sequence of the light-strand origin of replication

In the novel replication mechanism of closed circular mouse L-cell mitochondrial DNA synthesis one strand of the duplex (the heavy-strand) is initiated at a defined origin and proceeds unidirectionally. Synthesis of the complementary light-strand is initiated at a different origin, located approximately two-thirds genome length from the heavy-strand origin, and also proceeds unidirectionally. The initiation of light-strand synthesis does not occur until synthesis of the heavy-strand has extended past the light-strand origin region. One intriguing consequence of this asynchrony is that the heavy-strand origin functions in a DNA duplex, while the light-strand origin functions as a single-stranded template. In order to obtain the precise location of the light-strand origin we have isolated replicative molecules in which light-strand synthesis has begun and subjected them to digestion by a combination of the single-strand specific nuclease S₁ and various restriction cndonucleases. By comparison of the sizes of the duplex fragments thus generated with those produced by cleavage of non-replicating molecules cleaved with the same enzymes we have located the 5′-end of daughter light-strands at a position 55 to 90 nucleotides from a HpaI cleavage site 0.67 genome length from the heavy-strand origin. The nucleotide sequence of a 318-base region surrounding this site, determined by chemical sequencing techniques, possesses the symmetry required for the formation of three hairpin loops. The most striking of these has a stem consisting of 12 consecutive basepairs and a 13-base loop. In the heavy-strand template, this loop contains 11 consecutive thymidine nucleotides. This light-strand origin region has been found to possess a remarkable degree of homology with several other prokaryotic and eukaryotic origin-related sequences, particularly those of the øX174 A region and the simian virus 40 EcoRII G fragment.It has previously been shown that mouse mitochondrial DNA contains alkali-labile sites, which are presumably due to the presence of ribonucleotides incorporated into the DNA. A cluster of sites, representing eight adjacent ribonucleotides, has been located in mature light strands at or near the origin of light-strand synthesis. The retention of ribonucleotides at this specific location may reflect inefficient removal of an RNA primer at the light-strand origin.     

In vitro protein-DNA interactions at the human lamin B2 replication origin

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.     

In vitro initiation of DNA replication in simian virus 40 chromosomes

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.     

The initiation of SV40 DNA synthesis is not unique to the replication origin

Replicative intermediates of SV40 were isolated, digested with the restriction endonuclease Bgl I and examined by electron microscopy. Over 98% of the replicative intermediates isolated following infection with wild-type virions at 33 degrees, 37 degrees or 40 degrees C or with tsA209 at 33 degrees C had initiated replication about 35 nucleotides to one side of the Bgl I site. Approximately 1% of the molecules had initiated replication about 2400 nucleotides from the Bgl I site. The remaining molecules may have initiated at other sites. When tsA209 virion-infected cultures were shifted to 40.5 degrees C for 90 min, the relative rate of thymidine incorporation into superhelical viral DNA dropped by more than 97%. The remaining incorporation was not due to "leakiness." The label incorporated into mature superhelical molecules during brief pulses was not preferentially incorporated near the terminus of replication as it was at 33 degrees C. Approximately 33% of the incorporated label represented repair synthesis. Electron microscopy revealed that half of the replicative intermediates formed under these conditions appear to have been initiated randomly around the SV40 genome. Rolling circle molecules contaminated all the preparations of replicative intermediates.     

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.     

DNA replication origin interference increases the spacing between initiation events in human cells

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.     

Mechanism of mitochondrial DNA replication in mouse L-cells: RNA priming during the initiation of heavy-strand synthesis

The major form of mouse L-cell mitochondrial DNA contains a small displacement loop at the replication origin, created by synthesis of a 550 to 670-nucleotide portion of the heavy strand. These short heavy-strand segments remain hydrogen-bonded to the parental light strand and are collectively termed 7 S mitochondrial DNA. The unique location of these 7 S mitochondrial DNAs at the heavy-strand origin suggests that they may function as primers in the synthesis of full-length heavy strands. Ribonucleotides have been detected at the 5′-end of some of these molecules, which are most likely remnants of primer RNAs. Using 5′-end labeling in vitro, we have determined that these ribonucleotides occur at several discrete positions along the nucleotide sequence of the origin region, which suggests that there may be variability in the precise initiation point of RNA priming or in the location of the switchover from RNA priming to DNA synthesis. The length of 5′-end RNA was estimated by alkali treatment of mitochondrial DNA prior to end labeling. A range of one to ten ribonucleotides was hydrolyzed from the 5′-end of some 7 S mitochondrial DNA strands. This is the first evidence of RNA priming at a eukaryotic cell DNA replication origin.     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication.

Summary The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.     

Mechanism of mitochondrial DNA replication in mouse L-cells: kinetics of synthesis and turnover of the initiation sequence

Pulse-chase radioactive labeling experiments using thymidine kinase-plus mouse LA9 cells have shown that the 7 S mitochondrial DNA initiation sequence of mitochondrial DNA is synthesized and turned over at a faster rate than previously determined. These pulse-chase labeling experiments have also determined that the replication time of mouse LA9 cell mitochondrial DNA is one hour. The halflife of pulse-labeled 7 S mitochondrial DNA initiation sequences is approximately 70 minutes. This turnover is so rapid that at least 95% of the mitochondrial DNA initiation sequences synthesized are lost to turnover without acting as primers for expansion synthesis of the mitochondrial DNA heavy strand. The mechanism of 7 S mitochondrial DNA turnover does not lead to significant accumulation of free 7 S mitochondrial DNA single-strands within mitochondria. Resynthesis of the 7 S mitochondrial DNA initiation sequence is sufficiently rapid that the majority of mitochondrial DNA molecules are maintained as displacement loop molecules. Approximately 20% of all nucleotides polymerized into mitochondrial DNA are incorporated into the 7 S initiation sequences. The size of newly synthesized 7 S mitochondrial DNA strands varies from about 500 to 620 nucleotides. Several size classes are resolved by polyacrylamide/urea gel electrophoresis and each class has approximately the same turnover rate.Mouse LD cells maintain their mitochondrial DNA genomes as unicircular, head-to-tail dimers. Since a significant fraction of these unicircular dimers contain only one displacement loop, the size of the initiation sequence in such molecules should be twice as long if synthesis of the strand is limited by the free energy of superhelix formation. An identical array of size classes of 7 S strands is obtained from this cell line as compared to mouse LA9 cells. This indicates that the extent of 7 S mitochondrial DNA synthesis is most likely determined by a nucleotide sequence specific event.     

In vitro replication of DNA containing either the SV40 or the polyoma origin

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).     

In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.     

The excised leader of human cytochrome c oxidase subunit I mRNA which contains the origin of mitochondrial DNA light-strand synthesis accumulates in mitochondria and is polyadenylated.

We identified a polyadenylated RNA species which contains the origin of human mitochondrial DNA light-strand synthesis and the surrounding complementary sequences of the four light-strand-encoded tRNAs. This RNA (RNA 9L) is probably derived from the leader portion of RNA 6 which is excised during the formation of the mature cytochrome c oxidase subunit mRNA (RNA 9). The high degree of secondary structure of this RNA is presumably responsible for its anomalous electrophoretic behavior in denaturing polyacrylamide gels.     

Assembly of the replication initiation complex on SV40 origin DNA

The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase α/primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.