CDK phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of the Drosophila origin recognition complex

Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2 x cyclin E and Cdk1 x cyclin B in vitro. Such phosphorylation of DmORC by Cdk2 x cyclin E, but not by Cdk1 x cyclin B, requires an "RXL" motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.     

DNA binding properties of the adenovirus DNA replication priming protein pTP

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.     

Mutations in the heterochromatin protein 1 (HP1) hinge domain affect HP1 protein interactions and chromosomal distribution

Heterochromatin Protein 1 (HP1) is a conserved component of the highly compact chromatin found at centromeres and telomeres. A conserved feature of the protein is multiple phosphorylation. Hyper-phosphorylation of HP1 accompanies the assembly of cytologically distinct heterochromatin during early embryogenesis. Hypo-phosphorylated HP1 is associated with the DNA-binding activities of the origin recognition complex (ORC) and an HMG-like HP1/ORC-Associated Protein (HOAP). Perturbations in HP1 localization in pericentric and telomeric heterochromatin in mutants for Drosophila ORC2 and HOAP, respectively, indicate roles for these HP1 phosphoisoforms in heterochromatin assembly also. To elucidate the roles of hypo- and hyper-phosphophorylated HP1 in heterochromatin assembly, we have mutated consensus Protein Kinase-A phosphorylation sites in the HP1 hinge domain and examined the mutant proteins for distinct in vitro and in vivo activities. Mutations designed to mimic hyper-phosphorylation render the protein incapable of binding HOAP and the DmORC1 subunit but confer enhanced homo-dimerization and lysine 9-methylated histone H3-binding to the protein. Mutations rendering the protein unphosphorylatable, by contrast, do not affect homo-dimerization or binding to lysine 9-di-methylated histone H3, HOAP, or DmORC1 but do confer novel DmORC2-binding activity to the protein. This mutant protein is ectopically localized throughout the chromosomes when overexpressed in vivo in the presence of a full dose of DmORC2. This ectopic targeting is accompanied by ectopic targeting of lysine 9 tri-methylated histone H3. The distinct activities of these mutant proteins could reflect distinct roles for HP1 phosphoisoforms in heterochromatin structure and function.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.     

Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping

Hyperphosphorylation of the Drosophila melanogaster origin recognition complex (DmORC) by cyclin dependent kinases (CDKs) allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. Here we present single particle electron microscopy (EM) studies of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states. 3D image reconstructions show that nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation of DmORC does not induce meso-scale rearrangement of the ORC structure. To better understand ORC's role in origin remodeling, we performed atomic force microscopy (AFM) studies that show the contour length of a 688bp linear DNA fragment shortens by the equivalent of approximately 130bp upon ORC binding. This data, coupled with previous studies that showed a linking number change in circular DNA upon ORC binding, suggests that ORC may wrap the DNA in a manner akin to DnaA. Based on existing data and our structures, we propose a subunit arrangement for the AAA+ and winged helix domains, and in addition, speculate on a path of the 133bp of DNA around the ORC complex.     

Co-operative interactions between NFI and the adenovirus DNA binding protein at the adenovirus origin of replication.

The DNA-protein and protein-protein interactions proposed for the stability of nucleoprotein complexes at the origin of replication in prokaryotes are also thought to impart regulatory precision in eukaryotic DNA replication. This type of specificity can be observed, for example, during adenovirus DNA replication where efficient initiation requires that nuclear factor I (NFI) binds to the origin of DNA replication. Addition of purified NFI stimulates the initiation of adenovirus DNA replication in vitro in a reaction that is dependent on the concentration of the adenovirus DNA binding protein (DBP). However, the molecular basis for the synergistic action of NFI and DBP during replication is at present unknown. We report here that DBP increases the affinity of NFI for its binding site in the replication origin. DBP did not, however, increase the affinity of another eukaryotic sequence-specific DNA binding protein, EBP1, for its recognition site. Other single-stranded DNA binding proteins could not substitute for DBP in increasing NFI affinity for its binding site. In addition, DBP was found to alter the binding kinetics of NFI, both by increasing the rate of association and decreasing the rate of dissociation of NFI with the DNA template. The co-operativity between NFI and DBP was also demonstrated on another DNA template, a human NFI site (FIB2), suggesting that this interaction is of general occurrence and not restricted to the adenovirus origin of replication.     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.     

The binding of a carcinogen to the nucleosomal and non-nucleosomal regions of the simian virus 40 chromosome in vivo

The effect of chromatin structure on the binding of a chemical carcinogen to the genomic DNA was studied. The binding in vivo of the ultimate carcinogen, benzo-pyrene 7,8,-diol,-9,10-epoxide, to various regions of the SV40 chromosome was revealed by an immunological method. Particular attention was given to restriction fragments which include the origin of replication which is "non-nucleosomal" in a significant fraction of the chromosomes. The distribution of (+/-) trans-7,8-dihydrobenzo[alpha]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts was studied in 1) SV40 DNA modified in vitro to a level of 20 adducts/molecule, 2) DNA from SV40 chromosomes modified in vivo to a level of less than 1 adduct, and 3) DNA from only those chromosomes with an open origin of replication. In other experiments, the binding of BPDE to the origin region was compared to the binding to nucleosome core particle DNA from the viral chromosome. The origin region bound 1.7-fold more BPDE than core DNA, while linker DNA is 3-fold more modified than core DNA. However, the origin region was only about 20% more modified than any other region of the chromosome. We conclude that while the conformation of the DNA in chromatin has a slight effect on its accessibility to the carcinogen, the SV40 chromosome does not contain a particular "hot spot" which is preferentially modified by BPDE.     

Adenovirus DNA replication in vitro: site-directed mutagenesis of the nuclear factor I binding site of the Ad2 origin.

The template requirements for efficient adenovirus DNA replication were studied in vitro in a reconstituted system with cloned DNA fragments, containing the Ad2 origin region, as templates. Replication is enhanced by nuclear factor I, a cellular protein that binds specifically to the Ad2 origin. This stimulation is shown to be strongly dependent on the concentration of the adenovirus DNA binding protein. Using synthetic oligonucleotides we have constructed plasmids with base substitutions in the nuclear factor I binding region. Footprint analysis and competition filter binding studies show that two of the three small blocks of conserved nucleotides in this region are involved in the binding of nuclear factor I. The binding affinity can be influenced by the base composition of the degenerate region just outside these two blocks. In vitro initiation and DNA chain elongation experiments with the mutants demonstrate that binding of nuclear factor I to the Ad2 origin is necessary for stimulation. However, binding alone is not always sufficient since a mutation which only slightly disturbs binding is strongly impaired in stimulation of DNA replication by nuclear factor I.     

Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.

The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila ovary is a model for investigating metazoan replication. Evaluation of cis sequence organization and function suggests that chorion loci share attributes with other replicons and provides insights into metazoan origin structure. Moreover, recent results indicate that chorion origins respond to S-phase control, but escape mechanisms that inhibit other origins from firing more than once in a cell cycle. Several identified genes that mediate amplification are critical for the cell cycle control of replication initiation. It is likely that further genetic screens for mutations that disrupt amplification will identify the cadre of proteins associated with origins and the regulatory pathways that control their activity. Furthermore, the recent development of methods to detect amplification in situ has uncovered new aspects of its developmental control. Examining this control will reveal links between developmental pathways and the cell cycle machinery. Visualization of amplifying chorion genes with high resolution also represents an opportunity to evaluate the influence of nuclear and chromosome structure on origin activity. The study of chorion amplification in Drosophila, therefore, provides great potential for the genetic and molecular dissection of metazoan replication.     

Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site.

We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E. coli lac promoter DNA site for CAP (fragment 'LCAP'). We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay. Under standard conditions [( NaCl] = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP. The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP. Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs. LCAP is attributable to this difference in ion-pair formation. The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.     

Interaction between gene II protein and the DNA replication origin of bacteriophage f1

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.     

Sequence-independent DNA binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli

The DnaA protein specifically binds to the origin of chromosomal DNA replication and initiates DNA synthesis. In addition to this sequence-specific DNA binding, DnaA protein binds to DNA in a sequence-independent manner. We here compared the two DNA binding activities. Binding of ATP and ADP to DnaA inhibited the sequence-independent DNA binding, but not sequence-specific binding. Sequence-independent DNA binding, but not sequence-specific binding, required incubation at high temperatures. Mutations in the C-terminal domain affected the sequence-independent DNA binding activity less drastically than they did the sequence-specific binding. On the other hand, the mutant DnaA433, which has mutations in a membrane-binding domain (K327 to I344) was inert for sequence-independent binding, but could bind specifically to DNA. These results suggest that the two DNA binding activities involve different domains and perform different functions from each other in Escherichia coli cells.     

N-(2-chloroethyl)-N-nitrosoureas covalently bound to nonionic and monocationic lexitropsin dipeptides. Synthesis, DNA affinity binding characteristics, and reactions with 32P-end-labeled DNA

The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in 32P-end-labeled restriction fragments with methidiumpropyl-EDTA.Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using 32P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. The alkylation of DNA by Cl-ENU-lex was compared to that by N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), which has no DNA affinity binding properties. While all the Cl-ENU compounds generate DNA breaks as a consequence of the formation of N7-alkyl-guanine, the Cl-ENU-lex compounds induced, in a time- and dose-dependent fashion, intense DNA cleavage bands at adenine, cytosine, and thymine residues associated with affinity binding sites. These non-G cleavages induced by Cl-ENU-lex were inhibited by the coaddition of distamycin at concentrations that did not affect G alkylation break sites. CCNU, even at much higher concentrations, does not generate any similar detectable lesions at non-G sites. Therefore, linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion.     

DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.