Stability of coliphage lambda DNA replication initiator, the lambda O protein.
The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.
Wegrzyn
G
G
Department of Molecular Biology, University of Gdańsk, Poland.
Pawlowicz
A
A
Taylor
K
K
D010582:Bacteriophage lambda / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism*
D004279:DNA, Viral; C027812:O protein, Bacteriophage lambda; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D008715:Methionine; D011938:Rec A Recombinases