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M Schnos

Publications and source records attributed to M Schnos.

5 recordsLinked to original sources
PubMed2000

New insights into protein-DNA interactions obtained by electron microscopy.

The electron microscopic study of DNA-protein complexes can yield valuable information that is often not easily available by other methods. In this article we give a number of examples that were chosen to illustrate the utility of electron microscopy. Along with the strategy used are protocols that allow such experiments to be carried out. The first example employs the following strategy. Points of close proximity between nucleic acid and protein within a bacteriophage or virus are made permanent by crosslinking. Bacteriophage or virus are then partially disrupted so that individual components can be visualized. With bacteriophages, such experiments show which DNA end first enters the host on infection and therefore can in principle indicate which phage genes would be first available for transcription. This type of experiment can also show which DNA end is first to be encapsulated during formation of the bacteriophage. Information on direction of encapsulation and indirectly, direction of replication of the rolling circles that lead to concatermeric DNA to be encapsulated, can also be derived. Such experiments can additionally accurately define the degree of DNA permutation, if present, within a bacteriophage population. Finally, examples are shown for in vitro reactions involving DNA, RecA, RecO, RecF, RecR, and SSB that lead to a further understanding of recombinational repair. Additionally antibody-gold labeling is used to locate various proteins in such complexes.

Adsorption↗
PubMed1991

Daughter origins can be held together by O protein in phage lambda replicative intermediates.

When lambda replicative intermediates are incubated with initiator protein O, complex molecules are observed in which O interacts with both daughter origin segments and with growing points. In the simplest of these molecules it appears that daughter origins and growing points may all be bound together at a single point. When replicative intermediates are sequentially incubated with single-stranded binding protein and O protein, simpler structures are observed. In this case, both daughter origins are bound together by O protein. This result mimics that found when plasmid containing two tandem lambda origin sequences is reacted with O protein. In this case double origin binding produces a DNA loop. Double origin binding, as demonstrated in this investigation, creates the potential for topological domains which will have important effects on the ability of daughter origins to initiate replication.

Bacteriophage lambda↗
PubMed1989

DNA looping induced by bacteriophage lambda O protein: implications for formation of higher order structures at the lambda origin of replication.

A plasmid has been constructed, pOri2, which contains two lambda replication origin sequences separated by 1068 bp; both lambda sequences having the same orientation. When lambda initiation protein O 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 O protein complex. Length measurements show that the O protein binds at the expected positions of the lambda origin sequences and that the looped area represents the DNA segment between the two O protein binding domains. Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of O 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 O to two origins fixes the linkage number of the intervening DNA. The double origin binding ability of O may be diagnostic of the details of the reaction of O with a single origin sequence. A model is presented that rests on the assumption that O can produce microscopic looping between O protein binding sites within a single ori sequence.

Bacteriophage lambda↗
PubMed1988

Initiation protein induced helix destabilization at the lambda origin: a prepriming step in DNA replication.

The interaction of the lambda phage initiator protein, O, with the lambda origin sequence, ori, has been investigated. Binding of O, or its amino-terminal fragment, causes a major structural change within a 60 bp 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 lambda 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 lambda after O binding.

Bacteriophage lambda↗