Interactions between Phage λ Replication Proteins, λ DNA and Minicell Membrane

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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‐λ‐infected minicells the λ DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31–36], appeard in two peaks of different buoyant densities: as a membrane‐bound and a free λ DNA. The covalently‐closed‐circular form of λ DNA appeared exclusively in the membrane fraction.The λ‐coded proteins, synthesized in λ‐infected minicells, appeared in two major fractions: as membrane‐bound and as free proteins, and in one minor fraction, bound with free λ DNA. Neither λ 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 λ DNA.The efect of high ionic strength (KCl) and of detergents (Triton X‐100 and sarcosyl) on the binding of replication proteins with λ DNA and with the membrane was studied. The non‐ionic detergent, Triton X‐100 caused displacement of a part of λ DNA from the membrane to the free λ DNA peak; both γ replication proteins were bound with free γ DNA. The binding of the O protein with λ DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.

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