REPLICATION OF BACTERIOPHAGE M13 .2. ROLE OF REPLICATIVE FORMS IN SINGLE-STRAND SYNTHESIS

Sucrose-gradient sedimentation of DNA from cells pulse-labeled with tritiated thymidine at 130 minutes after M13 infection shows a flow of material from RF‡ ‡ Abbreviations used: RF, replicative form; RFI, the covalently closed double-stranded ring form; RFII, the double-stranded ring form containing one or more single-strand breaks; SS, single strand; TdR, thymine deoxyriboside. to progeny single strands. For pulses less than 40 seconds, most of the phagospecific DNA sediments at the rates of RFI and RFII. Pulses for 15 seconds or less show a third component sedimenting even more rapidly than RFI. All of the pulse label in this component is contained in linear viral strands, some of which sediment even more rapidly than circular viral DNA in alkaline sucrose gradients. The label in RFII is contained almost entirely in linear viral strands of unit length. The relative amount of label in RFII decreases with increasing pulse lengths up to about 180 seconds. After a lag of at least 40 seconds, label rapidly accumulates in progeny single strands; by five minutes the distribution of label is similar to that observed in long-term labeling with 70 to 80% of the label in single-stranded DNA. When pulse-labeling is followed by growth in unlabeled medium, most of the incorporated label is chased into progeny single strands. Label is found in both viral and complementary strands of RF only after long-term labeling from the time of infection. Labeled complementary strands of RF molecules are conserved during further growth in unlabeled medium while labeled viral strands are displaced from the RF as new viral strands are synthesized. © 1969.