Regulation of the switch from early to late bacteriophage lambda DNA replication

• There are two modes of bacteriophage lambda DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (theta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in type cells the replication starting from ori lambda proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from ori lambda is predominantly unidirectional. The regulation of directionality of replication from ori lambda is mediated by positive control of lambda p(R) promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the p(tet) promoter instead of p(R) was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional theta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional theta mechanism early after infection due to efficient transcriptional activation of ori lambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of lambda genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of ori lambda resulting in unidirectional theta replication followed by sigma type replication.

Nyckelord

bacteriophage lambda development

Escherichia coli DnaA protein

rolling-circle DNA replication

theta DNA replication

transcriptional activation of origin

agarose-gel electrophoresis

escherichia-coli

coliphage-lambda

phage-lambda

nucleoprotein structures

transcriptional activation

plasmid replication

initiator function

gene-function

protein

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