The capacity for initiation and subsequent chain elongation was examined in several DNA temperature sensitive mutants of Escherichia coli after the mutants had been held at nonpermissive temperature for approximately 1.5 generation equivalents and then returned to permissive temperature in the presence of chloramphenicol. The results obtained indicate that 4-5 sets of replication forks can be initiated after return to permissive temperature in the presence of chloramphenicol but the forks apparently become stalled and fail to complete chromosomal replication in the presence of chloramphenicol. In temperature reversible dnaA mutants, once the chloramphenicol is removed the forks appear to be able to resume replication at the nonpermissive temperature. The relationship between premature initiation and premature chain termination is discussed.
This paper deals with the conditions that are necessary for the acquisition and expression of initiation potential in dnaA temperature sensitive mutants after they have been held for periods of time at nonpermissive temperature and then returned to permissive temperature in the presence of chloramphenicol. The following conditions were found to be essential: (1) 40-60 min at nonpermissive temperature during which time protein synthesis must occur; this period must be followed by (2) return to permissive temperature under which conditions active dnaA product is present, and (3) protein synthesis must be blocked during the first 10-20 min immediately after return to permissive temperature (when initiation takes place). In order for expression of the initiation potential (4) the chloramphenicol must be removed to allow the progression of the replication forks which had been initiated to occur and (5) the recA+ phenotype appears to be required for acquisition or expression (or both) of the initiation potential.
When protein synthesis was blocked in temperature-sensitive deoxyribonucleic acid synthesis mutants of Escherichia coli at nonpermissive temperatures, it reduced the amount of apparent subsequent chain elongation to approximately half that observed in the mutants either at nonpermissive temperatures alone or when protein synthesis was blocked at the permissive temperature. Blocking protein synthesis at the nonpermissive temperatures for periods of 40 min caused the loss of ability to reinitiate deoxyribonucleic acid synthesis at the permissive temperature.
The synthesis and action of the dnaA product with respect to DNA initiation and the synthesis of DNA-binding proteins in Escherichia coli was examined. Results indicate that when dnaA product is irreversibly denatured and must be synthesized before initiation can occur, its synthesis and action appear to be complete approximately 30 min before initiation takes place. However, in mutants whose dnaA product is temperature reversible the action of the dnaA product appears to occur near the time of initiation. Examination of the DNA-binding proteins from the mutants suggests that a 53 kd protein, possibly the dnaA product, may be synthesized at the time of initiation under normal conditions at permissive temperature. The presence of active dnaA product appears to trigger the synthesis of a 60-65 kd protein which may be responsible for preventing another immediate initiation event.
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