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Interactions between phage lambda replication proteins, lambda DNA and minicell membrane
Abstract:
Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.
Insights
Gentle lysis and fractionation of minicells revealed that phage lambda DNA and replication proteins associate with membranes. Detergents disrupt these interactions, affecting DNA-protein binding and membrane association.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage lambda DNA replication initiation involves specific proteins.
- Minicells, lacking chromosomal DNA, are useful for studying plasmid replication and phage gene expression.
- Prereplicative DNA intermediates can be trapped and analyzed.
Purpose of the Study:
- To develop gentle methods for minicell lysis and lysate fractionation.
- To investigate the localization and binding of phage lambda DNA and replication proteins within infected minicells.
- To determine the effects of ionic strength and detergents on these interactions.
Main Methods:
- Gentle lysis of minicells using T4 lysozyme without detergents.
- Fractionation of lysates by equilibrium sedimentation in metrizamide density gradients.
- Analysis of DNA and protein distribution in membrane-bound and free fractions.
Main Results:
- Phage lambda DNA, in prereplicative complexes, existed as membrane-bound and free forms.
- Covalently-closed-circular lambda DNA was exclusively membrane-bound.
- Lambda replication proteins (O and P) were primarily membrane-associated or bound to free DNA, not free proteins.
- Triton X-100 displaced DNA from membranes and bound replication proteins to free DNA.
- Sarcosyl disrupted the O protein-DNA complex.
Conclusions:
- Phage lambda DNA replication initiation involves membrane association.
- Replication proteins O and P interact with both membranes and DNA.
- Detergents differentially affect DNA-membrane and DNA-protein interactions during replication.