Maria Schnös

Partial denaturation of thymine- and 5-bromouracil-containing λ DNA in alkali☆

An electron microscopic examination of λ DNA, partially denatured at high pH, shows that the sections which denature first are at the same positions as those previously observed for λ DNA partially denatured by heat. A similar study of BU†-labeled λ DNA shows the A·BU-rich regions to be situated at similar positions along the molecule to the A·T-rich regions in normal λ DNA, but it also shows that the A·BU-rich regions melt out at a lower pH. Details of a modification to the protein film technique are given in the Materials and Methods section.

Position of branch points in replicating λ DNA

Abstract Recent experiments by Ogawa, Tomizawa & Fuke (1968) have shown that it is possible to isolate intracellular λ DNA having a circular structure but with an extra length of DNA extending between two branch points. An attempt has been made to map the position of these branch points with respect to the point on the circular structure corresponding to the ends of the mature λ phage DNA molecule. If the circular branched molecules do, in fact, represent replicating DNA, then certain conclusions may be drawn concerning the starting point and direction of replication.

Starting point and direction of replication in P2 DNA

Abstract Intracellular phage DNA has been isolated shortly after infection of Escherichia coli with P2 bacteriophage. In addition to simple circular molecules, the intracellular DNA was found to contain single-branch circles (circular molecules containing an extra tail segment). On the basis of denaturation map studies, the position from which replication originates and the direction in which it proceeds has been determined for the initial round of DNA synthesis.

Initiation protein induced helix destabilization at the λ origin: A prepriming step in DNA replication

Summary The interaction of the λ phage initiator protein, O, withthe λ origin sequence, ori , has been investigated. Binding of O, or its amino-terminal fragment, causes a major structural change within a 60 by AT-rich region just to the right of the O-binding site. ATP or other molecular energy sources are not required. The modification, as assayed by nuclease sensitivity, is reduced when certain ori mutant sequences, which bind O but fail to replicate, are substituted for the wild-type sequence. The modification of DNA structure caused by the interaction of O is absolutely dependent on the presence of superhelical tension at the λ origin sequence, and has several properties consistent with a strand separation reaction. We propose that this modification is a fundamental prepriming event that is the first stage in initiation of bidirectional replication in λ after O binding.

Structure of branch points in replicating DNA: Presence of single-stranded connections in λ DNA branch points

Abstract Replicating λ DNA molecules can be isolated from Escherichia coli shortly after phage infection. During the first round of replication λ DNA has a double-branched circular structure and frequently single-strand connections are observed at the branch points. A study has been made of the single-strand connections to branch points to obtain information about the molecular fine structure at the growing point during replication. The results are consistent with models which allow replication along both DNA strands but which require DNA synthesis only in the 5′ to 3′ direction. A model is presented which requires two sites of DNA synthesis to be associated with each branch point.

Some morphological properties of P4 bacteriophage and P4 DNA

Abstract P4 phage morphology and P4 DNA lengths have been studied by electron microscopy. Although P4 phage appears to contain the relatively complex structures of P2 phage (head, tail with contractile sheath and tail fibers), it is unusual in that its DNA content and head diameter are very much less than for P2 phage. The tails of P4 and P2 appear to be indistinguishable. These findings are consistent with the fact that P4 requires a helper genome to undergo a complete lytic growth cycle.

DNA looping induced by bacteriophage λ O protein: Implications for formation of higher order structures at the gl origin of replication

Abstract A plasmid has been constructed, pOri2, which contains two λ replication origin sequences separated by 1068 bp; both λ sequences having the same orientation. When λ initiation protein 0 is reacted with linearized pOri2 and examined by electron microscopy it is found to contain a looped area in which two parts of the plasmid are bound together by the 0 protein complex. Length measurements show that the 0 protein binds at the expected positions of the λ origin sequences and that the looped area represents the DNA segment between the two 0 protein binding domains. Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of 0 protein is used. When looped molecules are reacted with psoralen, crosslinked by irradiation with uv light, and then denatured, it is found that the looped area is more thermostable than the rest of the molecule. This indicates that the DNA within the looped segment is torsionally constrained while that outside the loop is free to rotate and suggests that simultaneous binding of 0 to two origins fixes the linkage number of the intervening DNA. The double origin binding ability of 0 may be diagnostic of the details of the reaction of 0 with a single origin sequence. A model is presented that rests on the assumption that 0 can produce microscopic looping between 0 protein binding sites within a single on sequence.

Location of the “variable end” of Mu DNA within the bacteriophage particle

Abstract When bacteriophage Mu is subjected to mild formaldehyde cross-linking conditions and spread for electron microscopy, the phage heads lyse and one end of the released DNA is found attached to the proximal end of the phage tail. If spreading is performed at pH 11.0–11.2 the DNA exhibits regions of partial denaturation in characteristic locations along the DNA molecule. Examination of the partial denaturation patterns of Mu DNA-tail complexes for wild-type Mu and for the insertion mutant MuX reveals that the tail is attached to a unique end of the DNA. Comparison of denaturation maps of DNA-tail complexes with those of Mu DNA heteroduplexes containing G bubbles and split ends indicates that the tail is attached to the variable end of Mu DNA. Similar analysis of denaturation maps of Mu DNA partially ejected through the tail demonstrates that the variable end is also the first end to be ejected from the phage particle. These results support the hypothesis that the variable end is the last section of Mu DNA to be packaged and the first to be released upon injection.

Electron microscopic identification of supercoiled regions in complex DNA structures.

When intracellular lambda replicative intermediates (theta structures) are intercalated with psoralen and then irradiated with long wavelength ultraviolet light (u.v.), interstrand crosslinks are produced. After purification and denaturation of these theta structures, a global difference in denaturation can be observed by electron microscopy; parental sections are essentially native whereas daughter segments are highly denatured. This difference can be explained if parental sections are covalently continuous (and therefore able to supercoil) and daughter segments are not. Due to the higher thermal stability of supercoiled DNA, parental DNA will remain native while daughter sections will denature. Because these structures are crosslinked, the thermal treatment does not lead to dissociation of the highly denatured daughter strands. Experiments with simple negatively supercoiled plasmid circles support the above conclusions. When circles are crosslinked with psoralen-u.v. and then denatured, they remain native because of the higher thermal stability of covalently closed structures. If the circles are linearized before heating but after the psoralen-u.v. treatment, the thermal stability effect is eliminated and the molecules become highly denatured. In this case, however, the crosslinking density is found to be higher than in samples linearized before psoralen-u.v. treatment. This, therefore, shows that crosslinking density also reflects the superhelical state of the molecule at the time of psoralen-u.v. treatment. Two different properties can be used to discriminate between supercoiled and covalently discontinuous domains in complex DNA structures. First, supercoiled regions remain native while covalently discontinuous segments denature following a thermal treatment. This effect requires that covalent continuity exists up to and during the heating treatment. Second, because negative superhelicity enhances psoralen intercalation, crosslinking density is higher in these regions. Even if supercoiled domains are destroyed after the psoralen-u.v. treatment, the imprint of superhelicity is retained and can be recognized as a higher than normal crosslinking density.

Caffeine-induced re-initiation of phage λ DNA replication☆

Abstract A significant proportion of bacteriophage λ replicative intermediates undergo reinitiation at the λ origin when 2 m m -caffeine is present. This phenomenon is not observed in the absence of caffeine. Apart from re-initiation, no other obvious change is observed in the replicative intermediates. In view of the above facts, alternative explanations for the effect of caffeine on eukaryote replicating systems are discussed.