The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides.

Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Here we show that Xenopus RLF-M consists of all six members of the MCM/P1 protein family, XMcm2-XMcm7. The six MCM/P1 polypeptides co-eluted on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitated from purified RLF-M. Further fractionation separated RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provided RLF-M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing.

Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor.

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

The DNA replication licensing system.

The Xenopus cell free system has proved a good model system to study in vitro DNA replication and the mechanism preventing rereplication in a single cell cycle. Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor (RLF), which binds to the chromatin before the G1-S transition of the cell cycle and is displaced during replication. The nuclear envelope prevents rebinding of RLF and hence relicensing. Nuclear envelope breakdown at mitosis is required to allow another round of replication. Protein kinase inhibitors block licensing factor activity and arrest Xenopus extracts in a G2 like state. These kinase inhibitors have allowed the development of an in vitro assay leading to the biochemical purification of RLF components. RLF can be separated into RLF-B and RLF-M, the latter consisting of several members of the MCM/P1 class of replication proteins. In Xenopus as well as in many other eukaryotes, the binding of MCM/P1 proteins to chromatin before S phase is essential for replication to occur. The proteins are then displaced as replication proceeds. These changes in subnuclear distribution are reflected by changes in the phosphorylation status. MCM/P1 proteins do not bind to the DNA on their own but need RLF-B to be loaded onto the chromatin. Their cycling behaviour is reminiscent of the existence of a prereplicative complex at the origins of replication in yeast, suggesting that the licensing mechanism is ubiquitous in eukaryotes.

The role of MCM/P1 proteins in the licensing of DNA replication.

The DNA replication licensing system ensures that eukaryotic chromosomes replicate precisely once per cell cycle. A central component of the licensing system, RLF-M, has recently been shown to consist of a complex of Mcm/P1 proteins. This result allows us to integrate data about the MCM/P1 family obtained in different eukaryotes, ranging from yeast to man, into a general picture of the way that chromosome replication is controlled.

Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA-binding proteins.

We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A. This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction. The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity. Sedimentation analysis suggests that this polypeptide exists as a monomer under high and low salt conditions. Dhel II is able to unwind long stretches of DNA, but with decreased efficiency. Addition of Escherichia coli-like single-stranded DNA-binding proteins stimulates the unwinding activity at least 10-fold on substrates greater than 200 nucleotides. In particular, the mitochondrial single-stranded DNA-binding protein isolated from Drosophila embryos is able to stimulate unwinding by dhel II. These properties show that the helicase described is different from another Drosophila helicase dhel I; it has thus has been classified as dhel II.

Purification and characterisation of a DNA helicase, dheI I, from Drosophila melanogaster embryos.

We have purified a DNA helicase (dhel l) from early Drosophila embryos. dhel l co-purifies with the single-stranded DNA binding protein dRP-A over two purification steps, however, the proteins can be separated by their different native molecular weight, with dhel l activity co-sedimenting with a polypeptide of approximately 200 kDa and a sedimentation coefficient of 8.6 S. The enzyme needs ATP hydrolysis and divalent cations for displacement activity. It is very salt sensitive, having a Mg2+ optimum of 0.5 mM and being inhibited by NaCl concentration > 10 mM. Dhel l moves 5'-->3' on the DNA strand to which it is bound. Unwinding activity decreases with increasing length of the double-stranded region suggesting a distributive mode of action. However, addition of dRP-A to the displacement reaction stimulates the activity on substrates with >300 nucleotides double-stranded region suggesting a specific interaction between these two proteins.

Mitochondrial single-stranded DNA-binding protein from Drosophila embryos. Physical and biochemical characterization.

Using a stringent purification procedure on single-stranded DNA cellulose, we have isolated the mitochondrial single-stranded DNA-binding protein from Drosophila melanogaster embryos. Its identity is demonstrated by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial protein is immunologically and biochemically distinct from the previously characterized nuclear replication protein A from Drosophila (Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R. (1993) Biochemistry 32, 5257-5266; Marton, R. F., Thömmes, P., and Cotterill, S. (1994) FEBS Lett. 342, 139-144). It consists of a single polypeptide of 18 kDa, which is responsible for the DNA binding activity. Sedimentation analysis suggests that D. melanogaster mitochondrial single-stranded DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein binds to DNA in its single-stranded form with a strong preference over double-stranded DNA or RNA, and binds to polypyrimidines preferentially over polypurines. Drosophila mitochondrial single-stranded DNA-binding protein exhibits a greater affinity for long oligonucleotides as compared to short ones, yet does not show high cooperativity. Its binding site size, determined by competition studies and by fluorescence quenching, is approximately 17 nucleotides under low salt conditions, and increases in the presence of greater than 150 mM NaCl. The homogeneous protein stimulates the activity of mitochondrial DNA polymerase from D. melanogaster embryos, increasing dramatically the rate of initiation of DNA synthesis on a singly primed DNA template.

Synthesis of the tomato golden mosaic virus AL1, AL2, AL3 and AL4 proteins in vitro.

Transcripts derived from the leftward region of tomato golden mosaic virus DNA A were translated in wheat-germ and rabbit reticulocyte lysate systems. The largest protein (M(r) 40K) produced from transcripts encompassing open reading frame (ORF) AL1 was identified as the AL1 protein by immunoprecipitation with AL1-specific antibodies. However the main product was a protein of M(r) 10K, that was shown by in vitro mutagenesis to be the product of AL4, an ORF contained within AL1 DNA in a different reading frame. Translation of transcripts containing ORF AL2 or ORF AL3 gave the AL2 and AL3 proteins respectively; both proteins were also efficiently produced from transcripts containing both ORFs which overlap over about two-thirds of their length. Translation of a transcript containing the four ORFs gave all four proteins, consistent with a previous report that three of these (AL1, AL2 and AL3) can be translated from a single polycistronic RNA in transgenic tobacco plants. It is suggested that the leftward region of DNA A of the whitefly-transmitted geminiviruses may be expressed by two principal messenger RNAs, one encoding the AL1 and AL4 proteins and the other encoding the AL2 and AL3 proteins, and that the AL4 and AL3 proteins may be translated from the messenger RNAs by a leaky scanning mechanism.

Purification and characterisation of dRP-A: a single-stranded DNA binding protein from Drosophila melanogaster.

Replication protein A (RP-A) is an essential single-stranded DNA binding protein (SSB) involved in the initiation and elongation phases of eukaryotic DNA replication. It has the ability to bind single-stranded DNA extremely tightly and possesses a characteristic hetero-trimeric structure. Here we present a method for the purification of RP-A from Drosophila melanogaster embryos. Drosophila RP-A (dRP-A) has subunits of about 66, 31 and 8 kDa, in line with analogues from other species. It binds single-stranded DNA very tightly via the large subunit. The complete protein has at least a 10- to 20-fold preference for single-stranded DNA over double-stranded DNA and it appears that binding is only weakly co-operative. Band shift experiments suggest that it has an approximate site covering the size of 16 nucleotides or less, however, it shows a greater affinity for long oligonucleotides than for short ones. We also demonstrate that dRP-A can stimulate the activity of its homologous DNA polymerase alpha in excess of 20 fold. Analysis of the protein's abundance during embryo development indicates that it varies in a manner akin to other replication proteins.

Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic dianthovirus.

Mutant movement proteins of red clover necrotic mosaic dianthovirus (RCNMV), consisting of in-frame deletions or fusions with a maltose-binding protein, were produced in Escherichia coli using expression vectors. The ability of the mutant proteins to bind to ssRNA was tested by photochemical cross-linking and gel retardation. The results showed that the region between amino acids 181 and 225 of the RCNMV movement protein contains an ssRNA-binding domain.

TGMV replication protein AL1 preferentially binds to single-stranded DNA from the common region.

The AL1 protein of tomato golden mosaic virus (TGMV) is encoded by the viral DNA and has been shown to be essential for viral DNA replication. We have over-expressed the AL1 open reading frame in E. coli and purified the protein from bacterial extracts to near homogeneity. Using various different techniques we have studied the interaction of the AL1 protein with DNA. The AL1 protein is able to bind to DNA containing the common region of the viral genome, which can be demonstrated by photochemical cross-linking. Binding is 4-fold stronger to single-stranded than to double-stranded DNA. Antibodies against the AL1 protein can be used to precipitate the protein-DNA complex. The binding to single- and double-stranded DNA is specifically to the common region since a DNA fragment unrelated to TGMV is not shifted in a gel retardation assay.

Eukaryotic DNA helicases: essential enzymes for DNA transactions.

DNA in its double-stranded form is energetically favoured and therefore very stable. However, DNA is involved in metabolic events and thus has a continuous dynamic. Processes such as DNA replication, DNA repair, DNA recombination and transcription require that DNA occurs transiently in a single-stranded form. This status can be achieved by enzymes called DNA helicases. These enzymes have the power to melt the hydrogen bonds between the base pairs by using nucleoside 5'-triphosphate hydrolysis as an energy source. A variety of different DNA helicases have recently been identified from eukaryotic viruses and cells. We focus on the current knowledge of these DNA helicases and their possible function in DNA transactions.

Properties of the nuclear P1 protein, a mammalian homologue of the yeast Mcm3 replication protein.

Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.

Four different DNA helicases from calf thymus.

Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Thömmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.

DNA polymerase epsilon: in search of a function.

The current model of eukaryotic DNA replication involves the two DNA polymerases delta and alpha as the leading and lagging strand enzymes, respectively. A DNA polymerase first discovered in yeast has now been found in all eukaryotic cells and is termed DNA polymerase epsilon. In yeast, the gene for DNA polymerase epsilon has recently been found to be essential for viability, raising new questions about its functions.

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).

Eukaryotic DNA replication. Enzymes and proteins acting at the fork.

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.

Glutaminyl-tRNA synthetase as a component of the high-molecular weight complex of human aminoacyl-tRNA synthetases. An immunological study.

The human glutaminyl-tRNA synthetase is three times larger than the corresponding bacterial and twice as large as the yeast enzyme. It is possible that the additional sequences of the human glutaminyl-tRNA synthetase are required for the formation of the multienzyme complex which is known to include several of aminoacyl-tRNA synthetases in mammalian cells. To address this point we prepared antibodies against three regions of the human glutaminyl-tRNA synthetase, namely against its enzymatically important core region, and against two sections in its large C-terminal extension. In intact multienzyme complexes the core region was accessible to specific antibody binding. However, the C-terminal sections became available to specific antibody binding only when certain components of the multienzyme complex were either absent or degraded. These findings allow first conclusions as to the relative position of some components in the mammalian aminoacyl-tRNA synthetase complex.

Eukaryotic DNA helicases.

DNA is very stable in its double-stranded form. For many processes of DNA metabolism, such as replication, repair, recombination and transcription, the DNA has to be brought transiently into a single-stranded form. DNA helicases are enzymes capable of melting the hydrogen bonds of base pairs by using the energy of nucleoside-5'-triphosphate hydrolysis. This minireview focuses on the current knowledge of DNA helicases from eukaryotic cells.