Regulation of DNA synthesis and capacity for initiation in DNA temperature sensitive mutants of Escherichia coli

Abstract

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.

Gene transcription and chromosome replication in Escherichia coli

“…At 40ЊC, ongoing rounds of replication are completed, new rounds are not initiated, and potential for initiation of two subsequent rounds of replication has accumulated (2,6,10,11,15,19,31). Upon return of the cultures to 30ЊC, the first round of replication is initiated immediately and the second is initiated about 25 to 30 min later (2).…”

Discontinuity in DNA replication during expression of accumulated initiation potential in dnaA mutants of Escherichia coli

“…Although it is clear from work in this laboratory and others (6,7,18,22) that the first burst of [3H]thymidine uptake upon expression of initiation potential corresponded to initiation of chromosome replication, it cannot be assumed a priori that the subsequent decrease in uptake corresponded to a decrease in (or cessation of) elongation of that round of DNA replication, as we suggest. The following evidence supports this suggestion, however.…”

“…1 nonpermissive temperature would yield a capacity for initiation of one round of replication on all chromosomes in the culture, then the anticipated experimental result would be an initial burst of [3H]thymidine uptake at 25°C corresponding to synchronous initiation of the rounds of replication, followed C minutes later by termination of those rounds. If the cells gained capacity for more than one round of replication at 41°C, a second burst of uptake corresponding to a second round of initiation would be expected at about 50 min (6,13), but this would not explain the sudden decrease in rate of uptake which began at 20 min. The first wave of…”

“…635 VOL. 165,1986 on May 10, 2018 by guest http://jb.asm.org/ Downloaded from quence of chloramphenicol addition, which can slow or stop chain elongation (6,7).…”

“…When a culture of a dnaA mutant is incubated at a temperature intermediate between permissive and nonpermissive, initiation frequency is limited by dnaA gene product activity and, consequently, a potential for initiation of chromosome replication accumulates in the cells (4,6,7,12,13,18,22,25,28,29,33). This initiation potential can be expressed by shifting the culture to a more permissive temperature in the absence of protein synthesis (e.g., in the presence of chloramphenicol) or round of replication, the first initiation event is followed by additional initiation events, with the time interval between the first and second events being about 30 min at 25 to 30°C (6,7,13). The initiation events require RNA polymerase activity for expression (7,13,15,25,36) and, apparently, a critical chromosomal superhelicity which could be needed for a transcriptional step or for other steps in initiation or both (9)(10)(11)27).…”