Countermeasures to survive excessive chromosome replication in Escherichia coli

Abstract

In Escherichia coli, like all organisms, DNA replication is coordinated with cell cycle progression to ensure duplication of the genome prior to cell division. Chromosome replication is initiated from the replication origin, oriC, by the DnaA protein associated with ATP. Initiations take place once per cell cycle and in synchrony at all cellular origins. DnaA also binds ADP with similar affinity as ATP and in wild-type cells the majority of DnaA molecules are ADP bound. In cells where the DnaA/DnaA ratio increases or in cells where DnaA has increased access to oriC, premature initiations take place, often referred to as overinitiation. Overinitiating cells are generally characterized by their slow growth and in the most severe cases lethal accumulation of DNA strand breaks. Here, we review the different strategies adopted by E. coli to survive overinitiation. We propose a unifying model where all mutations that suppress overinitiation keep replication forks separated in time and, thereby, reduce the formation of strand breaks. One group of mutations does so by lowering the activity of oriC and/or DnaA to reduce the frequency of initiations to an acceptable level. In the other group of mutations, replication forks are kept apart by preventing formation of damages that would otherwise cause replication blocks, by allowing bypass of replication blocks and/or by slowing down replication forks. This group of suppressors restores viability despite excessive chromosome replication and provides new insights into mechanisms that safeguard DNA integrity.

Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication

“…It is perhaps less obvious why the over-initiation activity of oriC allADP remained compatible with host growth. Over-initiation is most lethal when closely spaced replication forks are combined with some problem, such as a DNA lesion, that results in stalled forks, because this increases the frequency of co-directional collisions ( 28 , 56 ). To avoid this, cells maintain a minimal inter-initiation interval, termed the eclipse period, which ensures some spacing between replication forks.…”

“…Cells that carry too much DnaA-ATP (such as hda null cells) also over-initiate, and this is further complicated by DnaA-ATP-induced repression of nrdA B ( 61 , 62 ), resulting in lower than normal nucleotide levels. Insufficient nucleotides could slow fork movement directly, or could make it more difficult to bypass DNA lesions caused by events such as oxidative stress ( 56 , 63 ). In contrast oriC allADP cells retain functional SeqA, and the mechanisms controlling cellular DnaA-ATP levels should be operating normally.…”

HdaB: a novel and conserved DnaA-related protein that targets the RIDA process to stimulate replication initiation

“…3), as anticipated if the target of HdaB is HdaA ( Fig. 4), one would expect that suppressors of this toxicity should carry mutations that reduce the levels of active DnaA and/or that reduce initiation frequency and/or that restore viability despite excessive chromosome replication 21 . To test if this was indeed the case, we isolated random transposon mutants that could bypass the toxicity associated with the expression of HdaB Ae from Pxyl on a medium copy number vector in C. crescentus (Fig.…”

“…The second suppressor mutant carried a transposon insertion in the recQ gene encoding a DNA helicase involved in DNA repair 25 . Interestingly, RIDA deficiencies in E. coli were shown to provoke cell death through the accumulation of DNA strand breaks connected with the accumulation of reactive oxygen species (ROS) 21,26,27 and RecQ has been shown to be necessary for such ROS-dependent death as also seen during the so-called "thymineless death" (TLD) process 28,29 . Then, we tested whether C. crescentus cells over-expressing HdaB proteins accumulated more double-strand breaks (DSB) than control cells using a RecA-CFP reporter expressed from the endogenous Pvan promoter and fluorescence microscopy.…”

“…2), indicating the presence of ssDNA zones/lesions on the chromosome prior to cell death 33 . Such regions can become substrates for the production of double-stranded DNA breaks (DSB) by reactive oxygen species (ROS) leading to cell death in bacteria 21,26 . Interestingly, such rapid death in response to replication defects is reminiscent of the so-called thymineless death (TLD) process that underlies the action of several bactericidal drugs such as trimethoprim or sulfamethoxazole 29,34,35 .…”