DNA arrested mutants of gene 59 of bacteriophage T4. II. Replicative intermediates

The abnormal DNA replication of bacteriophage T4 containing amber nonsense mutants in gene 59 (amC5, amHL628) has been studied. Zonal centrifugal analysis of the mutant intracellular DNA molecules isolated from a nonpermissive host, Escherichia coli B, reveals the following: (1) Parental mutant DNA as well as nascent mutant DNA can be recovered by sucrose gradient centrifugation as a fast sedimenting fraction (>1500 S) which is presumably a cell-membrane DNA replication complex. (2) At 2-3 min after initiation of viral DNA synthesis, a gradual accumulation of mutant DNA with a sedimentation coefficient of 63 S is observed. The molecular weight of this DNA is close to, or a little less than, that of a mature viral DNA, suggesting that less than one round of replication occurs before the complete arrest of DNA synthesis. (3) In contrast to what is found in wild-type infected cells, little or no long single-stranded DNA (concatemer) appears in mutant infected cells. Concatenated molecules of DNA, which have been proposed as intermediates in viral replication, are normally found in wild-type infected cells 7 min after infection. (4) Unlike the case of cells infected with mutants of gene 46 and 47, addition of chloramphenicol prevents premature release of the mutant DNA of gene 59 from the fast-sedimenting fraction (>1500 S).