Eclipse period without sequestration in Escherichia coli

Abstract

Summary The classical Meselson–Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy (15NH4+ and 13C6‐glucose) medium were shifted to light (14NH4+ and 12C6‐glucose) medium. The HH‐, HL‐ and LL‐DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene‐specific probes. The eclipse period, estimated from the kinetics of conversion of HH‐DNA to HL‐ and LL‐DNA, turned out to be 0.60 generation times for the wild‐type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

“…However, the subsequent initiations in seqA mutants are not runaway (uncontrolled) in nature, presumably due to slowing down of replication forks (the actual reason is still unknown) . Interestingly, in seqA mutants the eclipse period is reduced to a bare minimum-as if it were determined by segregation itselfsuggesting that origin sequestration by SeqA is the major eclipse factor (Olsson et al 2002(Olsson et al , 2003.In contrast to thymineless death (Sangurdekar et al 2010;Kuong and Kuzminov 2012), cells subjected to other conditions known to cause replication fork inhibition incur no known irreparable chromosomal damage (the mutants with elevated ori/ter ratio do grow more slowly), showing that elevated CRC = 6 can be safely handled and causes neither cell death nor inhibition of the cell cycle. Perhaps thymine starvation is an exception to its sensitivity to increased CRC, while the eclipse phenomenon can be thought of as merely a time measure that standardizes distance between consecutive replication forks when fork progression is slowed, as in seqA mutants (Olsson et al 2002).…”

“…Interestingly, in seqA mutants the eclipse period is reduced to a bare minimum-as if it were determined by segregation itselfsuggesting that origin sequestration by SeqA is the major eclipse factor (Olsson et al 2002(Olsson et al , 2003.…”

“…However, the subsequent initiations in seqA mutants are not runaway (uncontrolled) in nature, presumably due to slowing down of replication forks (the actual reason is still unknown) . Interestingly, in seqA mutants the eclipse period is reduced to a bare minimum-as if it were determined by segregation itselfsuggesting that origin sequestration by SeqA is the major eclipse factor (Olsson et al 2002(Olsson et al , 2003.…”

“…Thyminelimited E. coli cells have 100% viability, show normal growth rates, and divide on time, but their CRC increases to compensate for the slower-moving replication forks (Zaritsky et al 2006). The system that regulates these extra initiations is proposed to be the one that determines the "eclipse period," a cell cycle phase of enforced origin inactivity following each replication initiation, lasting 60% of the generation time (von Freiesleben et al 2000;Olsson et al 2002). By preventing closely spaced origin firing events, the eclipse phenomenon defines a minimal allowed distance between codirectional replication forks in the E. coli chromosome.…”

“…Perhaps thymine starvation is an exception to its sensitivity to increased CRC, while the eclipse phenomenon can be thought of as merely a time measure that standardizes distance between consecutive replication forks when fork progression is slowed, as in seqA mutants (Olsson et al 2002). On the other hand, E. coli mutants with increased CRC suffer from increased chromosomal fragmentation (Michel et al 1997;Rotman et al 2014), while in humans increased CRC is linked to chromosome rearrangements (Hastings et al 2009;Zhang et al 2009) and carcinogenesis (Hook et al 2007;Truong et al 2014), suggesting that increased CRC is an important factor of genome instability.…”