DEPENDENCE OF PERIODIC DNA SYNTHESIS ON PROTEIN SYNTHESIS: DELAY OF DNA SYNTHESIS IN THE EARLY CLEAVAGE STAGE OF SEA URCHIN EMBRYOS TREATED WITH CYCLOHEXIMIDE AND COLCHICINE

The effects of cycloheximide and colchicine on cleavage and syntheses of DNA and proteins in cleaving embryos of the sea urchin, Hemicentrotus pulcherrimus , were examined. Cycloheximide caused delay of cell division with prolongation of the streak stage. Both inhibitors also caused delay in initiation of DNA synthesis. The decrease in the rate and prolongation of the period of DNA synthesis caused by these inhibitors varied with their concentrations and the time of administration. Initiation of DNA synthesis was delayed when cycloheximide was added to suspensions of embryos between the time after preceding DNA synthesis terminated and a definite time before the predicted time of initiation of the next synthesis of DNA, except at the stage of pronuclear fusion. However, when the inhibitor was added after initiation of the synthesis, the latter proceeded normally. Addition of 10 m m cycloheximide immediately after fertilization or 2 m m cycloheximide 60 min before fertilization also delayed DNA synthesis at the stage of pronuclear fusion, indicating that synthesis at this stage also required prior protein synthesis. Colchicine had less inhibitory effect on protein synthesis, but greatly delayed initiation of DNA synthesis and prolonged its duration. These facts suggest that a definite amount of a particular protein must be synthesized and accumulated in each synthetic cycle before initiation of DNA synthesis.     

Characterization of the stimulatory effect of T4 gene 45 protein and the gene 4462 protein complex on DNA synthesis by T4 DNA polymerase

On a variety of single-stranded DNA templates, the overall rate of in vitro DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase is increased about fourfold by addition of the T4 gene $\text{44}\text{62}$ and 45 proteins. Several different methods suggest that this stimulation reflects an increase in the average DNA polymerase “sticking distance”, or processivity, from 800 to about 3000 nucleotides per initiation event. Both the $\text{44}\text{62}$ protein complex and the 45 protein must be present to obtain this effect, and either ATP or dATP hydrolysis is required. Rapid-mixing experiments indicate that the polymerase stimulation is maximized within a few seconds after addition of these “polymerase accessory proteins.”     

Conditional coupling of leading-strand and lagging-strand DNA synthesis at bacteriophage T4 replication forks

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.     

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.     

Deletion of sites for initiation of DNA synthesis in the origin of broad host-range plasmid R1162

The origin of replication of the broad host-range plasmid R1162 contains two, oppositely facing initiation sites for DNA synthesis. Either of these sites can be deleted from an R1162 plasmid derivative. However, the resulting plasmids are unstable, maintained at a lower copy-number in the cell, and form dimers and other recombinants that are required for propagation of the plasmid. In vitro, a derivative lacking one initiation site is deficient in synthesis of the strand normally initiated from that site. The properties of the intact origin are restored if it contains two oppositely facing sites; one initiation site may substitute for the other, and each site need not be in its original orientation. Overall, the results suggest that synthesis of each strand of R1162 DNA is initiated at a single site, and that there is no efficient system for initiation of lagging strand synthesis during transit of the replication forks.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Dependence of timing of mitotic events on the rate of protein synthesis and DNA replication in sea urchin early cleavages

Abstract. To understand what processes affect the cell-cycle timing of mitotic events in early cleavage cycles of sea urchin embryos, a study was made on the effects of (a) reducing protein synthesis with emetine and (b) DNA replication with aphidi-colin, on the timing of nuclear envelope breakdown, anaphase onset and cytokinesis. When protein synthesis was slightly inhibited by administration of emetine, the delay in the mitotic events increased, with an increase in the delay in accumulation of proteins up to the levels to which cells must synthesize the proteins to execute the cleavage. This indicated that protein synthesis affects the timing of mitotic events. The delay in cleavage cycles caused by a slight inhibition of DNA replication with aphidicolin was in proportion to the concentration of aphidicolin administered, suggesting that DNA replication also affects the timing of mitotic events. Furthermore, it was confirmed that accumulation of the proteins to the levels required for execution of the first cleavage precedes completion of DNA replication as a requirement for execution of the first cleavage. These results imply the existence of process(es) affected by protein synthesis that are included in a feedback control system which prevents the initiation of mitosis until after the completion of DNA replication; it is the characteristic of a cell-cycle control system that has been predicted theoretically.     

Role of individual monomers of a dimeric initiator protein in the initiation and termination of plasmid rolling circle replication

Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.     

Studies of the DNA helicase-RNA primase unit from bacteriophage T4. A trinucleotide sequence on the DNA template starts RNA primer synthesis

The purified DNA replication proteins encoded by genes 41 and 61 of bacteriophage T4 catalyze efficient RNA primer synthesis on a single-stranded DNA template. In the presence of additional T4 replication proteins, we demonstrate that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for RNA primer-dependent initiation of new DNA chains. These chains start with primers that have the sequences pppApCpNpNpN and pppGpCpNpNpN, where N can be any one of the four ribonucleotides. Each primer is initiated from the T (A-start primers) or C (G-start primers) in the center of the recognized template sequence. A subset of the DNA chain starts is observed when one of the four ribonucleoside triphosphates used as the substrates for primer synthesis is omitted; the starts observed reveal that both pentaribonucleotide and tetraribonucleotide primers can be used for efficient initiation of new DNA chains, whereas primers that are only 3 nucleotides long are inactive. It was known previously that, when 61 protein is present in catalytic amounts, the 41 and 61 proteins are both required for observing RNA primer synthesis. However, by raising the concentration of the 61 protein to a much higher level, a substantial amount of RNA-primed DNA synthesis is obtained in the absence of 41 protein. The DNA chains made are initiated by primers that seem to be identical to those made when both 41 and 61 proteins are present; however, only those template sites containing the 5'-GCT-3' sequence are utilized. The 61 protein is, therefore, the RNA primase, whereas the 41 protein should be viewed as a DNA helicase that is required (presumably via a 41/61 complex) for efficient primase recognition of both the 5'-GCT-3' and 5'-GTT-3' DNA template sequences.     

Distinctive features of the respiratory syncytial virus priming loop compared to other non-segmented negative strand RNA viruses

De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation.     

Gene Expression and Stability of mRNA Affected by DNA-Arrested Synthesis in Gene 59, 46, and 47 Mutants of Bacteriophage T4

The effect of bacteriophage T4 gene 59 mutations (DNA-arrested synthesis) on kinetics of DNA synthesis, gene expression, and stability of mRNA has been studied. When Escherichia coli B was infected by a T4 gene 59 mutant, DNA synthesis proceeded to increase linearly after initiation, but started to decrease at 8 min and was completely arrested at 12 min at 37°C. At various incubation temperatures (20 to 42°C), the initial rates and times of arrest of DNA synthesis were different, but the total amount of DNA synthesized was constant. This result supports the hypothesis that function of gene 59 is required for the conversion of 63S DNA molecules to other replicative intermediates (39). The abnormality in protein synthesis caused by gene 59 mutation is manifested by (i) a delayed shutoff in the expression of early proteins (gene 43, 46, 39, 52, 63, 42-45, and some unidentified proteins), (ii) a reduced rate of late gene expression (gene 34, 37, 18, 20, 23, wac, 24, 22, 38, and 19), and (iii) an absence of cleavage of certain late proteins (23, 24, IPIII and 22 to 23^*, 24^*, IPIII^*, and small fragments). It appears that there was no effect on the expression of gene 33, 55, and 32 by a mutation in gene 59. Results obtained from an addition of rifampin at the prereplicative cycle after infection indicated that mRNA from genes 43, rIIA, 46, 39, 52, and 63 are more stable in T4amC5 (gene 59) than in wild-type-infected cells. mRNA remained functional longer in mutant-infected cells, and this may explain the prolonged synthesis of certain early proteins. The gene expression of other DNA arrested mutants—those in genes 46 and 47—showed a pattern of abnormal protein synthesis similar to that found in gene 59 mutant-infected cells, except more late proteins are synthesized. The gene expression in terms of phage DNA structure is discussed.     

Regulation of DNA synthesis and capacity for initiation in DNA temperature mutants of Escherichia coli. III. Synthesis of the dnaA protein and of DNA-binding proteins

Summary The synthesis and action of the dnaA product with respect to DNA initiation and the synthesis of DNA-binding proteins in Escherichia coli was examined. Results indicate that when dnaA product is irreversibly denatured and must be synthesized before initiation can occur, its synthesis and action appear to be complete approximately 30 min before initiation takes place. However, in mutants whose dnaA product is temperature reversible the action of the dnaA product appears to occur near the time of initiation. Examination of the DNA-binding proteins from the mutants suggests that a 53 kd protein, possibly the dnaA product, may be synthesized at the time of initiation under normal conditions at permissive temperature. The presence of active dnaA product appears to trigger the synthesis of a 60–65 kd protein which may be responsible for preventing another immediate initiation event.     

Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis

Four steps are known to be required for the initiation of DNA synthesis in Saccharomyces cerevisiae. Three of these are mediated by the products of genes cdc 4, 7, and 28 and the fourth is identified by the inhibition exerted on haploid α cells by the mating pheromone, α factor. These four steps have been ordered by a combination of two methods and found to be:

initiation of DNA synthesis The two sequencing methods are described in detail. Experiments involving the shift of mutant cells from the restrictive to the permissive temperature in the presence of cycloheximide demonstrated that the protein synthesis requirement for yeast DNA replication can be completed before the cdc 7-mediated step.     

Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes

A crude soluble enzyme system capable of authentic replication of a variety of oriC plasmids has been replaced by purified proteins constituting three functional classes: initiation proteins (RNA polymerase, dnaA protein, gyrase) that recognize the oriC sequence and presumably prime the leading strand of the replication fork; replication proteins (DNA polymerase III holoenzyme, single-strand binding protein, primosomal proteins) that sustain progress of the replication fork; and specificity proteins (topoisomerase I, RNAase H₁ protein HU) that suppress initiation of replication at sequences other than oriC, coated with dnaA protein. Protein HU and unidentified factors in crude enzyme fractions stimulate replication at one or more stages. Replication has been separated temporally and physically into successive stages of RNA synthesis and DNA synthesis.     

Single-molecule insight into stalled replication fork rescue in Escherichia coli

DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.