Tests of spool models for DNA packaging in phage lambda

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine.In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map.In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with ³²P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found.These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on λ DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982).A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into λ phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method. Sites of AMT crosslinking appear duplex; uncrosslinked regions appear as single-stranded loops. AMT is found to intercalate throughout the λ DNA. Patterns of reacted sites appear different from one DNA molecule to the next, and no consistent pattern can be found. More extensive intercalation occurs with the deletion mutant λb221 than with phage of wild-type DNA length, and free DNA shows much more reaction than the DNA inside either phage type. In order for intercalation to occur, the DNA helix must unwind and become further extended. This experiment shows that regions throughout the entire DNA molecule can unwind and be extended by intercalation, which is not confined to a single DNA segment or to segments in one half of the DNA molecule, as would be expected for the two spool models if only the DNA in contact with the capsid were accessible to the dye.     

Crosslinking of the complementary strands of DNA by UV light: dependence on the oligonucleotide composition of the UV irradiated DNA

UV light crosslinks the complementary strands of DNA. The interstrand crosslinks may contribute to the biological and pathological effects that UV irradiation is known to bring about. Here alkaline agarose gel electrophoresis was used to assess the crosslinked fraction of 31 selected restriction fragments of six viral and plasmid DNA molecules exposed to UVC light irradiation. As many as 17 independent experiments were performed with the particular DNA fragments to get sufficiently precise data suitable for quantitative analyses. The data were used to determine how the crosslinked fraction depended on the dinucleotide, trinucleotide and tetranucleotide contents of the irradiated DNA fragments. This analysis demonstrated that DNA conformation and/or flexibility, rather than the local double helix thermostability, governed the phenomenon of crosslinking. For example, (GA).(TC) suppressed the crosslink formation in DNA more than any dinucleotide composed of only G and C. In addition, (CTAG).(CTAG) promoted crosslinking much more than any other tetranucleotide, including e.g. (TATA).(TATA), whereas the closely related (CATG).(CATG) belonged among the tetranucleotides that most suppressed the UV light induced crosslinks between the complementary strands of DNA. The present data reproduced crosslinking of the analyzed 31 restriction fragments with a correlation coefficient exceeding 0.90. This result will be useful to predict crosslinking along the whole human genome.     

Studies on the arrangement of DNA inside viruses using a breakable bis-psoralen crosslinker

We have developed a new DNA-DNA crosslinking strategy based on a cleavable bispsoralen reagent and used this strategy to study the structures of bacteriophage lambda and the animal virus SV40. Our results show that in both lambda and SV40, all restriction fragments examined can be crosslinked to all other restriction fragments. In bacteriophage lambda, the crosslinking data are consistent with a random packaging model, while in SV40 there is some deviation from the random model. These results imply that the structures of DNA inside these viruses are either highly disordered or very complex.     

Structure of the Escherichia coli 16 S ribosomal RNA. Psoralen crosslinks and N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine crosslinks detected by electron microscopy

Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule.     

Primary organization of the nucleosome core particles sequential arrangement of histones along DNA

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particles has been determined by a new sequencing procedure. The lysine groups of histones were crosslinked to partly depurinated DNA at neutral pH. One strand of DNA was split at the points of crosslinking, thus leaving the 5′-terminal DNA fragments bound to histones. The lengths of these crosslinked DNA fragments were measured to determine the position of histones on one strand of the core DNA from its 5′ end.The results demonstrate that histones are bound to regularly arranged, discrete DNA segments about six nucleotides long. These segments are separated by histone-free gaps about four nucleotides wide located at a distance of about 10n nucleotides from the 5′ end of DNA. The first 20 nucleotides from the 5′ ends of DNA seem to be free of histones. Histones appear to be arranged symmetrically and in a similar way on both DNA strands. Any one histone, being bound predominantly to discrete segments on one or other of the strands, can oscillate at the same time between the two strands across the major DNA groove. Two symmetrical models for the arrangement of two molecules of each core histone on linearized and folded DNA are proposed.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.     

HindII, HindIII,and HpaI restriction fragment maps of the left arm of bacteriophage λ DNA

The sites on the left arm of bacteriophage λ DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII+III), and Hemophilus parainfluenzae (HpaI) were localized on the λ physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis. The restriction sites within the b2 region of λ were mapped by analysis of the digestion profiles of deletion and substitution derivatives of λ, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. The restriction sites on the λ genome between the left vegetative end and the b2 region were mapped entirely by successive digestion experiments. The restriction fragment map for the right arm of λ may be found in the accompanying paper (Robinson and Landy, 1977).     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). I. Undamaged covalent circular lambda DNA molecules in uvrA+ recA+ lysogenic host cells are cut following superinfection with psoralen-damaged lambda phages.

When λ bacteriophages were treated with a photosensitizing agent, psoralen or khellin, and 360 nm light, monoadducts and interstrand crosslinks were produced in the phage DNA. The DNA from the treated phages was injected normally into Escherichia coli uvrA^? (λ) cells and it was converted to the covalent circular form in yields similar to those obtained in experiments with undamaged λ phages. In excision-proficient host cells, however, there was a dose-dependent reduction in the yield of rapidly sedimenting molecules, and a corresponding increase in slow sedimenting material, the extent of this conversion corresponding to about one cut per two crosslinks. Presumably, the damaged λ DNA molecules were cut by the uvrA endonuclease of the host cell, but were not restored to the original covalent circular form.The presence of psoralen damage in λ phage DNA greatly increased the frequency of genetic exchanges in λ phage-prophage crosses in homoimmune lysogens (Lin et al., 1977). As genetic recombination is thought to depend on cutting and joining in DNA molecules, experiments were performed to test whether psoralen-damaged λ DNA would cause other λ DNA in the same cell to be cut. E. coli (λ) host cells were infected with ³²P-labeled λ phages and incubated to permit the labeled DNA to form covalent circles. When these host cells were superinfected with untreated λ phages, there was no effect upon the circular DNA. When superinfected with λ phages that had been treated with psoralen and light, however, many of the covalent circular molecules were cut. The cutting of undamaged molecules in response to the damaged DNA was referred to as “cutting in trans”. It required the uvrA⁺ and recA⁺ host gene functions, but neither recB⁺ nor any phage gene functions. It occurred normally in non-lysogenic hosts treated with chloramphenicol before infection. Cutting in trans may be one of the steps in recA-controlled recombination between psoralen crosslinked phage λ DNA and its homologs.     

Analysis of EDTA-chelatable proteins from DNA-protein crosslinks induced by a carcinogenic chromium(VI) in cultured intact human cells

DNA-protein crosslinks (DPCs) were induced in intact human leukemic T-lymphocyte MOLT4 cells or isolated nuclei by treatment with potassium chromate, chromium(III) chloride hexahydrate or x-rays. The proteins complexed to DNA were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE). A group of identical non-histone proteins was crosslinked to DNA by any of the three treatments, except that a 51 kDa basic protein was additionally complexed to DNA when either potassium chromate or chromium(III) chloride hexahydrate was the crosslinking agent. Treatment of chromate-induced DNA-protein crosslinks with EDTA or thiourea followed by ultracentifugation dissociated the major proteins from the complex indicating that these proteins were crosslinked to DNA by direct participation of a EDTA-chelatable form of chromium such as Cr(III) through sulfur containing amino acid residues. The 51 kDa protein was not seen in the post-EDTA pellet but was present in the post-thiourea pellet, indicating that it was also crosslinked to DNA by Cr(III) through non-sulfur-containing amino acids. Digestion of x-rays-induced DPCs by DNase I also revealed this protein on two-dimensional gels indicating that the same protein was also crosslinked by oxidative mechanisms. The involvement of oxidative mechanisms in the crosslinking process was indicated as the majority of the proteins in chromate-induced DPCs were resistant to EDTA and thiourea treatment, and were found to crosslink to DNA when x-rays were used as the crosslinking agent. These results suggest that the chromate-induced DPCs are formed by the generation of reactive oxygen species during the intracellular chromate reduction as well as by the biologically generated Cr(III). About 19% of DNA-protein crosslinks actually involve Cr(III) crosslinking DNA to proteins, about 14% involve Cr(III) crosslinking DNA to proteins through non-sulfhydryl containing moieties and about 5% involve Cr(III) crosslinking DNA to sulfhydryl groups on proteins. The remaining 81% of DNA-protein crosslinks appear to be oxidatively crosslinked out of which about 45% appear to be through sulfhydryl groups and another 36% appear to be through non-sulfhydryl groups.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution.

Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg²⁺, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution.     

Superhelical Escherichia coli DNA: relaxation by coumermycin.

A class of compounds of the form: NH₂NHCO(CH₂)_nC(OR)₂CHR′X has been designed to allow selective blocking of specific genetic sequences of DNA and RNA (Summerton, unpublished data). This paper describes the synthesis and use of 6-bromo-5,5-dimethoxyhexanohydrazide for such site-specific inactivation. In model reactions it is shown that this compound can be attached to the C-4 position of cytidine and that, after activation, the cytidine-bound agent crosslinks to the N-7 position of guanosine. This reaction sequence has been applied to the crosslinking of bacteriophage T7 RNA to its complementary DNA in a highly specific fashion. The RNA is derivatized with the hydrazide reagent, activated, and incubated under annealing conditions with the complementary DNA, resulting in crosslinks between the two strands that are stable to denaturing conditions, dependent on the presence of the crosslinking agent, and specific for the complementary DNA sequence. These studies show that the title compound is a promising sequence-specific blocking agent for nucleic acids. The capability of introducing site-specific blocks in DNA and RNA in this way may have a wide variety of applications in the study of genetic processes. In particular, the combination of this compound with appropriate restriction fragments may enable systematic mapping and characterization of viral genomes.     

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>10⁸ cts/min per μg) in vitro using deoxynucleoside [α-³²P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.     

Genomic DNA regions whose complementary strands are prone to UV light-induced crosslinking

We prepared an extensive set of DNA restriction fragments, irradiated them with UV light, and detected crosslinked complementary strands by electrophoresis in denaturing agarose gels. These experimental data were quantified by densitometry to determine tetranucleotide contributions to crosslinking. The tetranucleotide contributions were used to predict genomic maps of the crosslinking probability that permitted us to identify two strongly crosslinking genomic regions having 295 and 389 base pairs in length. The two sequences shared the (ATTTTATA).(TATAAAAT) octamer, which is a candidate for the hotspot of UV light-induced crosslinking between the complementary strands of DNA.     

Xpf and Not the Fanconi Anaemia Proteins or Rev3 Accounts for the Extreme Resistance to Cisplatin in Dictyostelium discoideum

Organisms like Dictyostelium discoideum, often referred to as DNA damage “extremophiles”, can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other “extremophiles” can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage–resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents.     

Cloning of the yeast ribosomal DNA repeat unit in SstI and HindIII lambda vectors using genetic and physical size selections.

The size of DNA fragments complementary to ribosomal RNA was determined in SstI and HindIII restriction spectra from totally and partially cleaved yeast (Saccharomyces cerevisiae) DNA. The results indicated that the yeast ribosomal RNA gene cluster consists of 9000 base-pair long tandemly repeated units. Three different repeating units, which are overlapping with respect to their sequences, were cloned as SstI and HindIII fragments with λ vectors. The isolation of these clones was facilitated by genetic or physical preselection for those recombinant phage which contained DNA inserts in the expected size range. Both preselection methods gave about a 30-fold purification with respect to the λ-rDNA clones. A heteroduplex analysis of the clones obtained with a three-component HindIII vector showed that the center part of the λ genome carrying λ recombination and regulation genes (57 to 77% λ) can become inverted without apparent decrease of growth capacities.     

Terminal fragments of herpes simplex virus DNA produced by restriction endonuclease.

Digestion of HSV-1 DNA with λ 5′-exonuclease prior to digesting the DNA with the $\text{Eco R}$ I restriction endonuclease specifically affects two of the fragments normally obtained after restriction endonuclease digestion. Therefore these two fragments contain the sequences which occur at the termini of HSV-1 DNA. One of the fragments affected is a “minor” fragment which is always present in less than molar yield. The possible relationship between the occurrence of minor $\text{Eco R}$ I fragments and the partial refractoriness of HSV-1 DNA to λ 5′-exonuclease digestion is discussed.     

Physical mapping of bacteriophage lambda DNA with restriction endonuclease HpaI

The restriction endonuclease from Haemophilus parainfluenzae, endoR·HpaI cleaves λcI857s7 DNA into 14 fragments. The sizes of these fragments were determined and a physical map was constructed. The ordering of the fragments was carried out using different deletion and substitution mutants of λ phage, double cleavages with another restriction enzyme, endoR·BamHI, and partial protection of individual HpaI recognition sites by the antibiotics distamycin A and actinomycin D. HpaI produces fragments from the left arm of the λ DNA genome, which may help in investigating the structure and function of this part of the phage.     

Studies of DNA packaging into the heads of bacteriophage lambda

During the assembly of bacteriophage λ heads, a head-like, DNA-free structure called petite λ, is first constructed. Into this, λ DNA is then packaged. In this paper we examine early interactions between λ DNA and petite λ in a cell-free system. The two major findings of this paper are: (1) when seen through the electron microscope, an early petite λ-λ DNA complex appears with the circular petite λ having the DNA crossing through its center. These resemble a bead on a string or the Greek letter φ (hence they are called φ structures). The λ A protein is required in the formation of φ structures. Also, φ structures can be found in bacteria infected with phage λ. (2) The polyamine putrescine is required for phage head assembly. An earlier reported requirement for spermidine can be replaced by the addition of putrescine. Polyamine is required in the DNA packaging reaction after the packaging has begun.