The mechanism of replication of øX174 DNA. XIII. Discontinuous synthesis of the complementary strand in an escherichia coli host with a temperature-sensitive polynucleotide ligase

Escherichia coli KS252, isolated by Konrad et al. (1973), carries a thermosensitive ligase by virtue of the lig ts7 mutation and is capable of supporting limited growth of bacteriophage øX174 at the restrictive temperature. Analysis of intermediates in øX DNA replication synthesized at 41°C yielded the following results.o(a)During the synthesis of the parental replicative form (RF) most of the [³H]thymidine incorporated into DNA after a short pulse appeared in 5 S to 9 S fragments as analysed by velocity sedimentation in alkaline sucrose. In vivo these fragments appeared to be precursors of full-length DNA; in vitro, as part of a replicative form molecule, they could be joined together by polynucleotide ligase.(b)The [³H]thymidine incorporated during a 30-s labelling period during RF replication was found in polydeoxynucleotide strands ranging from 5 S to 9 S to longer than unit-length. Competition annealing experiments revealed that the unit-length and longer øX174 DNA comprised mostly viral strand sequences while the 5 S to 9 S fragments were composed exclusively of complementary strand sequences. Although some of the viral strand DNA labelled by a 5-s pulse was longer than unit-length, most of it was unit-length. In replicating intermediates, viral strand sequences were found in part to be single-stranded, suggesting that synthesis of the viral strand preceded synthesis of the complementary strand.(c)During single-stranded DNA synthesis, no 5 S to 9 S DNA was found and much of the pulse-labelled DNA was longer than unit-length. We conclude that synthesis of the complementary strand, but not of the viral strand, of øX174 is obligatorily discontinuous and that the discontinuities found in the nascent DNA in the lig ts7 mutant are repaired by the E. coli NAD-dependent polynucleotide ligase.