Peculiarities of the secondary structure of bacteriophage DNA in situ: IV. Covalent cross-links between DNA and protein that arise in the reaction of S d phage with O-methylhydroxylamine

Abstract

S d phage were incubated in 1 m- O-methylhydroxylamine. At various time-intervals, samples of modified phage were isolated and disrupted either by heating or by treatment with detergent. Changes in viscosity and buoyant density of disrupted preparations took place in the course of modification. Three transient synchronous drops in viscosity and buoyant density levels were observed with minima at five minutes, one and three hours of modification. The specific viscosity of the preparations at minima was 10 to 20% that of the disrupted unmodified phage. Properties of the phage preparation isolated during the third period of decreased viscosity were studied in more detail. This preparation, subjected to thermal disruption, gives a single DNA-containing band in Cs 2SO 4 gradient centrifugation corresponding to a buoyant density of 1.37 g/cm 3 (cf. 1.39, 1.29 and 1.43 g/cm 3 for whole phage, phage ghosts and native phage DNA, respectively). The band contains practically all the 35S label that was present in the starting phage, suggesting that it corresponds to a complex of phage DNA with protein. Electron microscopy revealed complexes as thick strands of 50 to 300 Å diameter bonded to globular particles of varying size. In four hours of modification, the viscosity and buoyant density of disrupted phage returned to values characteristic of unmodified preparations. The DNA band contained no 35S label. Electron microscopy of the substance of this band revealed fibres of 20 Å diameter. A possible explanation of the results is based on the assumption of pre-existing non-covalent interaction of C (4)—NH 2 moieties of cytidine residues with nucleophilic groupings of coating protein within the virion. It is assumed that it is this interaction that holds DNA in “non-native” conformation within intact phage particles and thus explains its peculiar properties discovered earlier. In the present case, the interaction determines the formation of DNA-protein crosslinks under O-methylhydroxylamine treatment via the earlier postulated intermediate product of cytosine modification. Restoration of “normal” physical properties of disrupted phage after more prolonged modification is explained by cleavage of the DNA-protein cross-links due to reaction of the postulated intermediate with O-methylhydroxylamine affording N (4)-methoxy-6-methoxy-amino-5,6-dihydrocytidine residues.