Insufficient levels of the nrdAB‐encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain

Abstract

The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and β clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA (RIDA). In addition to hyper-initiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., ΔseqA or ΔdatA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δhda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.

Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome

“…It was shown before that deletion of rrn operons affects the growth rate of cells only moderately [ 38 , 39 ]. However, in a recent study, a duplication of a similar location was observed as a suppressor of the severe growth defect of cells lacking the DnaA regulatory inactivator Hda [ 40 ]. The suppression of the slow-growth phenotype of Δhda cells was found to be the increased gene dosage for DNA polymerase I ( polA ) [ 40 ].…”

“…However, in a recent study, a duplication of a similar location was observed as a suppressor of the severe growth defect of cells lacking the DnaA regulatory inactivator Hda [ 40 ]. The suppression of the slow-growth phenotype of Δhda cells was found to be the increased gene dosage for DNA polymerase I ( polA ) [ 40 ]. This or other similar effects might be important contributors in oriX cells.…”

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

“…SeqA null mutants over-initiate, and because the closely spaced forks encounter unrepaired collapsed forks, growth of SeqA null cells is severely inhibited. 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 ).…”

Coping with Reactive Oxygen Species to Ensure Genome Stability in Escherichia coli

“…These mutations affect iron/sulfur cluster synthesis, Flavin/Fe reduction, ArcA and/or respiration [ 82 , 84 ]. In the hyper-replicating cells, the gaps generated by MutM are toxic [ 10 ] and aerobic growth can be restored in the absence of MutM [ 10 ] or when PolI is overproduced [ 85 ]. Recombination repair functions are essential under these conditions [ 86 ] and the DNA damage seen in hyper-replicating cells is avoided in absence of oxygen.…”