Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

Cell cycle inhibition of yeast spheroplasts

Summary Osmotically stabilized yeast spheroplasts are capable of extensive DNA synthesis. Although the rate of DNA synthesis in spheroplasts is approximately one-third that of intact cells, the relative amounts of nuclear and mitochondrial DNA synthesized by spheroplasts is very similar to the relative amounts synthesized by intact cells. Furthermore, nuclear but not mitochondrial DNA synthesis is inhibited in MATa spheroplasts by the application of the yeast mating pheromone, -factor. Similarly, DNA synthesis is reversibly temperature-sensitive in spheroplasts created from cdc7 and cdc8 mutant cells.     

Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication.

The replication of the 2 μm DNA of Saccharomyces cerevisiae has been examined in cell division cycle (cdc) mutants. The 2 μm DNA does not replicate at the restrictive temperature in cells bearing the cdc28, cdc4, and cdc7 mutations which prevent passage of cells from the G1 phase into S phase. Plasmid replication also is prevented in a mating-type cells by α factor, a mating hormone which prevents cells from completing an event early in G1 phase. The 2 μm DNA ceases replication at 36 °C in a mutant harboring the cdc8 mutation, a defect in the elongation reactions of nuclear DNA replication. Plasmid replication continues at the restrictive temperature for approximately one generation in a cdc13 mutant defective in nuclear division. These results show that 2 μm DNA replication is controlled by the same genes that control the initiation and completion of nuclear DNA replication.     

Sequential function of gene products relative to DNA synthesis in the yeast cell cycle

An experimental rationale for deciphering the relative dependence of steps in a developmental pathway (Jarvik & Botstein, 1973; Hereford & Hartwell, 1974) has been employed to determine the relationship between the hydroxyurea-sensitive step and various temperature-sensitive steps in the cell cycle of Saccharomyces cerevisiae. Since hydroxyurea inhibits DNA replication in yeast (Slater, 1973), the data identify gene products upon whose function DNA replication is dependent (cdc 4, 6, 7, 2, 8, 21) and gene products whose function or synthesis requires DNA replication (cdc 2, 8, 21, 9, 13, 16, 23, 5, 15). Other gene products (cdc 3, 11, 24) function independent of DNA replication. These results suggest that the events of the cell cycle occur in a proscribed order because many of the gene products that mediate these events arc restricted to a prescribed sequence of function.Mutations in two genes (cdc 2 and 6) result in cells that remain sensitive to hydroxyurea after an incubation at the restrictive temperature, despite the fact that both mutants incorporate radioactive precursors into DNA at the restrictive temperature (Hartwell, 1973). It is suggested that cdc 6 specifies a function that is necessary for the proper initiation of DNA replication, and cdc 2 a function that is necessary for correct DNA elongation, and that in the absence of either of these functions the DNA that is made is either faulty or incomplete.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

Genetic control of the cell division cycle in yeast : II. Genes controlling DNA replication and its initiation

Temperature-sensitive mutations occurring in two unlinked complementation groups, cdc4 and cdc8, are recessive and result in a defect in DNA replication at the restrictive temperature. Results obtained with synchronous cultures suggest that cdc4 functions in the initiation of DNA replication and cdc8 functions in the propagation of DNA replication.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe

Summary A total of 59 new temperature sensitive cdc mutants are described which grow normally at 25°C but become blocked at DNA replication or mitosis when incubated at 36°C. Thirtynine of the mutants are altered in cdc genes which have been identified previously. The remaining 20 mutants define 10 new cdc genes. These have been characterised physiologically, and 6 of the genes (cdc 17, 20, 21, 22, 23, 24) were found to be required for DNA replication, 2 for mitosis (cdc 27, 28), and 2 (cdc 18, 19), could not be unambigously assigned to either DNA replication or mitosis but were definitely required for one or the other.Three genes, the previously identified cdc 10, and cdc 20, 22 are likely to be required for the initiation of DNA replication. Mutants in two genes, cdc 17, 24 undergo bulk DNA synthesis at 36°C, but this DNA is defective. In the case of cdc 17 the defect is in the ligation of Okazaki fragments. cdc 23 is required for bulk DNA synthesis, whilst cdc 21 may possibly be required for the initiation of a particular sub-set of replicons.A previously isolated mutant cdc 13.117 is also further described. This mutant becomes blocked in the middle of mitosis with apparently condensed chromosomes.     

A probe into nuclear events during the cell cycle of Saccharomyces cerevisiae: studies of folded chromosomes in cdc mutants which arrest in G1

The sedimentation behavior of folded chromosomes from celldivision-cycle (cdc) mutants which arrest in g ₁ was examined. At the restrictive temperature the folded genome of cdc 7, which arrests after spindle pole body (SPB) separation and spindle formation, cosediments with a standard g ₁ structure, indicating that by the cdc 7 step the g ₁ form of the folded genome has been assembled. In the mutant, cdc 4, which arrests before SPB separation but after SPB duplication, a standard g ₁ structure is not formed, cdc 4 cells, however, are able to enter G₀ at the restrictive temperature, and the corresponding g ₀ structure is stable. These results indicate that the cdc 4 gene product may be involved in the development of folded genome conformation which leads to the g ₁ structure. Since the cdc 4 gene product is required for SPB separation, the g ₁ structure may be defined by an association between chromosomes and spindle components. The folded chromosomes of the start mutants cdc 25 and cdc 28 are unstable at the restrictive temperature. In contrast to cdc 4, neither cdc 25 nor cdc 28 are able to enter the G₀ stage in a normal manner, i.e., the g ₀ structure is unstable at the restrictive temperature. The inference is that both the cdc 25 and cdc 28 gene products are required for the functional integrity of the folded genome at both a stage early in G₁ and in the pathway to G₀.     

A DNA Polymerase-����Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

α-Accessory factor (AAF) stimulates the activity of DNA polymerase-α·primase, the only enzyme known to initiate DNA replication in eukaryotic cells (Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265 ,13221 -13230). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44·AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-α·primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-³H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-α·primase template affinity and stimulate both DNA primase and polymerase-α activities in vitro.In eukaryotic cells, DNA replication is initiated at multiple origins internal to each chromosome; the origin recognition complex recruits cell division cycle and minichromosome maintenance proteins to form a preinitiation complex (1). At the G₁-S phase transition, the latter complex is activated by cyclin-dependent protein kinases leading to formation of an initiation complex that alters local DNA structure through DNA helicase activity (1, 2). The replication protein A (RPA)² is recruited to bind and stabilize single-stranded DNA (ssDNA) produced by the initiation complex (3, 4). RPA serves as an auxiliary factor for DNA polymerase-α (pol-α)·primase: it stabilizes the protein complex by direct interaction with both pol-α and primase subunits, and it reduces the misincorporation rate of pol-α, acting as a “fidelity clamp” (5, 6). The pol-α·primase complex consists of four subunits, including the catalytic pol-α subunit (p185), a regulatory B subunit (p70), and two primase subunits (p49 and p58). On an ssDNA template, the primase synthesizes short RNA primers from ribonucleoside triphosphates (rNTPs), which are elongated by pol-α in the presence of deoxyribonucleoside triphosphates (dNTPs) to form short DNA fragments. Through mechanisms requiring other replication factors, pol-α·primase is replaced by the more processive DNA polymerases pol-δ and pol-ε (7). Pol-ε synthesizes the leading strand, whereas pol-δ completes each Okazaki fragment initiated by pol-α·primase on the lagging strand and proofreads errors made by pol-α (7). The initiator RNA and DNA fragments are later removed by nucleases, and the Okazaki fragments are sealed by DNA ligase (7).The pol-α·primase complex is the only eukaryotic DNA polymerase able to initiate DNA synthesis de novo. In addition to initiating DNA replication and synthesizing Okazaki fragments, it appears to be one of the final targets of cell cycle checkpoint pathways that couple DNA replication to DNA damage response (2, 8). The role of RPA in initiation, elongation, and completion of lagging strand DNA synthesis has been thoroughly investigated (3, 9), but in vitro studies suggest that some additional factors that promote the rapidity of DNA replication in vivo are still lacking (2).In the course of purifying pol-α·primase from extracts of cultured mouse L1210 cells, we identified a factor we named α-accessory factor (AAF) that stimulates pol-α·primase activity in vitro (10, 11). The protein has a native molecular mass of ∼150 kDa as determined from its sedimentation coefficient and Stokes radius and is composed of two subunits of ∼132 and ∼44 kDa. AAF stimulates pol-α·primase activity with several different templates and types of reactions: (i) It stimulates selfprimed reactions with poly(dT), poly(dI·dT), or single-stranded circular DNA; (ii) it stimulates primed reactions with poly(dA)·oligo(dT) and multiply primed DNA in the absence of rNTPs, indicating that it affects pol-α activity when no primers are being made; and (iii) it stimulates primase activity on ssDNA in the absence of dNTPs, showing that it can enhance RNA primer synthesis in the absence of DNA synthesis (11). AAF increases the template affinity and processivity of pol-α·primase (12). AAF is highly specific for pol-α·primase and has no effect on the other mammalian DNA polymerases β, γ, or δ or on the DNA polymerase·primase complexes from Drosophila and Saccharomyces cerevisiae (11).The cloning of both AAF subunits based on peptide sequences obtained from the purified protein allowed us now to further characterize the AAF-44·AAF-132 complex structurally and functionally. Based on siRNA experiments in cancer cell lines, AAF appears to regulate DNA replication in vivo.     

Initiation of DNA Synthesis in HeLa Cell-free System

THE molecular mechanism for initiating DNA replication can be studied using a subcellular system. Rao and Johnson¹ found that HeLa cells in the pre-DNA-synthetic (G-1) period of the cell cycle initiate DNA synthesis after fusion with cells that are in the DNA synthetic (S) period. A previous subcellular system of DNA replication from HeLa cells^2–4 consisted of intact nuclei, supplemented with the four deoxy-nucleoside triphosphates, salt, ATP and a cytosol factor. The nuclei in this system appeared to be permeable to proteins and DNA synthesis was very similar to that within intact cells. We report here the initiation of DNA synthesis in nuclei isolated from HeLa cells. Our results suggest that, with the synchronization method used, a small percentage of dormant G-1 nuclei can be stimulated by S-phase cytoplasm; this would be the case if the cells were receptive to stimulation for only 30–60 min during the cell cycle. illustration

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A DNA sequence cleaved by restriction endonuclease R.EcoRI in only one strand.

Restriction endonuclease EcoRI cuts both strands of the DNA sequence

generating two separate frayed ends (Hedgpeth et al., 1972). Here it is shown that under standard digestion conditions, the enzyme also attacks the sequence

but cuts only one strand. The resulting nick is an efficient initiation point for DNA synthesis by Escherichia coli DNA polymerase I, allowing the selective labelling of one strand of the DNA duplex.In buffers of low molarity and high pH (8.5), EcoRI cleaves sequences with the form

(Polisky et al., 1975). Thus it seems that under both sets of conditions the enzyme recognises the four-base-pair core sequence

and that its ability to cleave different adjacent phosphodiester bonds varies with pH and ionic strength.     

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg⁴⁰⁴, Lys⁴⁰⁸, Tyr⁴¹¹, and Gly⁴¹⁵) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr⁴¹¹ and Gly⁴¹⁵ are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp²⁶³ located in the linker of one subunit can interact with Tyr⁴¹¹, Lys⁴⁰⁸, or Arg⁴⁰⁴ in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp²⁶³ and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.At the replication fork DNA helicase unwinds the duplex DNA to expose single-stranded DNA for use as templates for the leading and lagging strand DNA polymerases (1). The 5′ to 3′ polymerization of nucleotides by the leading strand DNA polymerase proceeds in a continuous manner, whereas synthesis on the lagging strand occurs in a discontinuous manner, generating Okazaki fragments. The synthesis of each Okazaki fragment is initiated by the extension of an oligoribonucleotide that serves as a primer for the lagging strand DNA polymerase. These oligoribonucleotides are synthesized in a template-directed manner by DNA primase. For the two polymerases to communicate with each other, the lagging strand folds back on itself such that the lagging strand DNA polymerase becomes part of the replisome. This association of the two polymerases enables both strands to be synthesized in the same overall direction, and synthesis of both strands proceeds at identical rates. The folding of the lagging strand creates a replication loop of lagging strand DNA that contains the nascent Okazaki fragment and the ssDNA³ extruded behind the helicase. Single-stranded DNA-binding protein binds to the exposed single-stranded DNA to remove secondary structure, but it also interacts with the other proteins of the replisome to assist in the coordination of DNA synthesis (2).Among the several protein interactions within the replisome, the interaction of the helicase with the primase is one of the most critical (2–6). The association of the primase with the helicase places it in position to catalyze primer synthesis on the single-stranded DNA extruded by the moving helicase. In addition, the higher affinity of the helicase for single-stranded DNA serves to stabilize the primase on the lagging strand. Perhaps the most important is the ability of the primase to communicate with the helicase. During the rate-limiting step of primer synthesis, leading strand synthesis would be expected to outpace lagging strand synthesis. The association of primase with helicase provides a mechanism by which helicase movement can be coordinated with primer synthesis (7).The gene 4 protein of bacteriophage T7 is unique in that it contains both helicase and primase activities within the same polypeptide chain (see Fig. 1A). Although separate genes encode other replicative helicases and primases, they nonetheless require a physical association to function properly (2, 5). The helicase activity resides in the C-terminal 295 residues, and the primase activity resides in the N-terminal 245 residues (8). A linker of 26 residues separates the helicase and primase domains. The linker plays a critical role in the oligomerization of gene 4 protein (9). The primase and the helicase domains have been purified separately and shown to exhibit their activities independently (9–11). However, the presence of each domain has striking effects on the activity of the other (2).Open in a separate windowFIGURE 1.Elements involved in the interaction between helicase and primase in E. coli and bacteriophage T7. A, schematic presentation of helicase and primase together with the structural elements involved in their interaction. In E. coli the helicase and primase interact via contacts of the C-terminal p16 of the primase with the N-terminal p17 of the helicase. In bacteriophage T7 the two activities are found in a single polypeptide where the primase and helicase domains are covalently connected via a flexible linker. Helix E is located in the helicase domain. B, top view of the hexameric T7 helicase (right panel) (Protein Data Bank accession code 1E0J). C, side view of the heptameric gene 4 protein containing both the helicase and primase domains (right panel) (Protein Data Bank accession code 1Q57). In B and C, two adjacent subunits are shown in green and yellow, respectively. The linker region and residues Ala²²⁵–Gly²²⁶ in the primase domain of the green subunit are shown in blue and magenta, respectively. Helix E in the helicase domain of the adjacent yellow subunit is shown in red. Residues potentially involved in the in trans interaction at the interface are indicated (left panels). In the heptameric structure (C), Gly⁴¹⁵ in Helix E is potentially interacting with Ala²²⁵ and/or Gly²²⁶ from the primase domain of the adjacent subunit. Lys⁴⁰⁸ is close to Asp²⁶³ in the linker from the adjacent subunit. In the hexameric structure (B), because the primase domain and a portion of the linker region are missing in this structure, the counterpart of Gly⁴¹⁵ is not present. Another obvious difference in this structure compared with that shown in C is that Asp²⁶³ in the linker of the heptamer is oriented toward Lys⁴⁰⁸, whereas it is close to Tyr⁴¹¹ in the hexamer structure. Distances shown in B and C are in similar ranges regardless of locations of interfaces in both hexameric and heptameric gene 4 protein structures. Structures from the Protein Data Bank were analyzed using PyMOL (DeLano Scientific LLC).Like other members of the Family 4 helicases, the helicase domain of gene 4 protein functions as a hexamer (see Fig. 1B). Members of this family assemble on single-stranded DNA with the DNA passing through the central channel formed by the oligomerization (4, 12). The nucleotide-binding site of the helicase is located at the subunit interface located between two RecA-like subdomains that bind dTTP, the preferred nucleotide for T7 helicase (13–17). The location of the nucleotide-binding site at the subunit interface provides multiple interactions of residues with the bound dTTP (18). These interactions assist in oligomerization, in binding to DNA, and in coupling the hydrolysis of dTTP to mechanical movement of the helicase (19–23).The primase domain, residing in the N-terminal half of the gene 4 protein, is a member of the DnaG family of prokaryotic primases. Three structural features distinguish members of this family. An N-terminal zinc-binding domain plays a critical role in recognizing sites for primer synthesis in ssDNA. An RNA polymerase domain, linked to the zinc-binding domain by a flexible linker, contains the catalytic site where metal-dependent polymerization of nucleotides occurs. A C-terminal segment covalently attaches the primase to the helicase. In other primases of this family, this segment interacts with the cognate helicase. T7 primase, like the primases of phage T4 and Escherichia coli, recognizes a trinucleotide sequence (5). T7 primase recognizes the sequence 5′-GTC-3′, at which it catalyzes the template directed synthesis of a dinucleotide (pppAC); the 3′-cytosine is essential for recognition, although this “cryptic” nucleotide is not copied into the product (24). The dinucleotide is then extended by the primase, provided the proper nucleotides, T and G, are present in the template. Consequently, the predominant T7 primase recognition sites are 5′-GGGTC-3′, 5′-TGGTC-3′, and 5′-GTGTC-3′ (25, 26). Thus T7 primase catalyzes the synthesis of the tetraribonucleotides pppACCC, pppACCA, and pppACAC. The lagging strand DNA polymerase then extends these functional tetranucleotides.The covalent linkage of primase and helicase in the gene 4 protein of bacteriophage T7 distinguishes it from most other replication systems where the association of the two proteins is dependent on a physical interaction of the two separate proteins. In bacteria such as E. coli, Bacillus stearothermophilus, and Staphylococcus aureus, this interaction is mediated through two structurally similar regions: the helicase-binding domain (p16 domain) located at the C terminus of the DnaG primase and the p17 domain of the DnaB helicase located at the N terminus of the protein (see Fig. 1A) (27–31). The association of DnaB with DnaG alters sequence recognition by DnaG and affects the length of primers synthesized (28, 31–33). Furthermore, cooperative binding of two or three DnaG monomers to the hexameric DnaB can halt translocation of DnaB on DNA (34). Such “association and dissociation” between the helicase and primase mediated by the p16 and p17 domains are believed to coordinate DNA synthesis by regulating the initiation of Okazaki fragment synthesis (6, 35, 36). Mutations in the p16 domain of DnaG can either affect the ability of the two proteins to form a complex, enhance the primase activity, or modulate the ATPase and/or helicase activities allosterically (31).The covalent association of primase and helicase in the bacteriophage T7 system clearly provides several of the advantages derived from the physical association of the two proteins in other systems. The primase is positioned correctly for primer synthesis, and DNA binding is achieved via the helicase. Furthermore, communication between the two domains of the gene 4 protein is dramatically revealed by the cessation of helicase movement during primer synthesis (7). However, the covalent association of the two activities precludes regulation by dissociation as in the other replication systems. The frequency of primase recognition sites in the phage genome is considerably more than that required for the initiation of Okazaki fragments. Consequently, primase activity in the T7 replication system must be highly regulated to ensure the translocation of helicase and the almost constant length of Okazaki fragments (2).T7 gene 4 protein is present in solution as a mixture of hexamers and heptamers (37), and the crystal structures of both oligomeric forms have been determined (see Fig. 1, B and C) (15, 38). In the heptameric structure an interaction of the helicase and primase domains occurs through helix E (see Fig. 1C). Located at the front of the helicase domain facing toward the primase domain, helix E is not only in proximity to the primase of the adjacent subunit but also in contact with the linker region connecting the two domains of the adjacent subunit. By this trans-packing interaction, the primase domain from one subunit is loosely stacked on the top of the helicase from the adjacent subunit (38) (see Fig. 1C). In the six-membered ring structure (15), the functional form of gene 4 protein, the primase domain is missing. However, the contact between helix E and the linker region from the adjacent subunit is present (see Fig. 1B). Some residues in the linker region have been identified previously as key factors involved in the conformational switch of helicase (39).How does the primase domain of gene 4 protein communicate with the helicase domain? Although the two domains cannot dissociate into solution, a transient dissociation of the two domains is possible as a result of the flexible linker through which they are connected. Alternatively, primase activity or helicase activity may be conveyed to the other domain as a result of conformational changes in the protein at the interface between the two domains. In either instance the linker region and the interface between the two domains are certain to be critical for this communication. Helix E, although quite distant from the catalytic sites of either the helicase or primase, contacts both the primase domain and the linker. In the present study we have examined the role of helix E in the function of gene 4 by genetically altering several residues and examining the function of the altered proteins in vivo and in vitro.     

The effect of the cdc9 mutation on premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

A diploid homozygous for cdc9, a conditional mutation defective in DNA ligase [2], has been used to investigate the role of this enzyme in premeiotic DNA synthesis. The cdc9 ligase has the same effect on premeiotic as on mitotic DNA synthesis and at the restrictive temperature the newly synthesized DNA is recovered in small fragments. A difference has been observed, however, between meiotic and mitotic cells, namely in their ability to join together these fragments on return to the permissive temperature. In mitotic cells this can be readly demonstrated within 50 min, whereas in contrast little joining was detected in meiotic cells, even after 2 h at the permissive temperature.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.