Testing models of the arrangement of DNA inside bacteriophage λ by crosslinking the packaged DNA

Abstract

Bis-psoralens can make crosslinks between two adjacent segments of a condensed DNA molecule. We have used bis-psoralen crosslinking as a covalent means of preserving structural features of DNA packaged inside bacteriophage λ. A single bis-crosslink prevents normal electron microscopic spreading of intact λ DNA: after deproteinization the molecules appear as tangled rosettes which are presumably due either to trapped knots or supercoils. However, restriction nuclease digestion of the crosslinked DNA yields fragments that spread normally. The location of crosslinks can be studied by their appearance in such a digest as X-shaped molecular features. Significant crosslinking frequencies are found between all six possible pairs of the four largest BglII fragments of λ DNA. Little or no evidence is seen for crosslinked loops within individual fragments. These results are inconsistent with two previously suggested models of intraphage DNA packaging. Determination of the positions of crosslinks within restriction fragments yields a pattern of DNA contacts too complex for any simple analysis. The finding of hints of periodicity in the sites of crosslinks, preferential crosslinking of some restriction fragments, and the occurrence of one particularly efficient crosslinking reaction between two restriction fragments appear to rule out purely random packaging arrangements.