Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant

Abstract

The dnaX gene of Escherichia coli encodes t and c clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in the dnaA gene that partly suppressed the dnaX2016 temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and t or c is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.

Overlap of replication rounds disturbs the progression of replicating forks in a ribonucleotide reductase mutant of Escherichia coli

“…2d, Table 1). This effect has been previously described by other groups using dnaA defective alleles alone (Gon et al, 2006) or combined with dnaX alleles that impaired the progression of DNA replication (Skovgaard & Løbner-Olesen, 2005).…”

Relationship between Fork Progression and Initiation of Chromosome Replication in E. coli

“…According to the model where the RNR is a component of the replication hyperstructure (Guzmán et al, 2002), it is reasonable to think that the defective fork progression observed in this mutant can be alleviated by reducing the number of replication forks running along the chromosome. Consistent with this, the presence of dnaA defective alleles, dnaA(Sx), suppresses the detrimental effect on DNA replication observed in mutants that have problems with the progression of forks due to the presence of defective subunits of DNA polymerase III coded by the dnaX gene (Gines-Candelaria et al, 1995;Blinkova et al, 2003;Skovgaard & Lobner-Olesen 2005). Furthermore, a lower availability of wild type DnaA protein induced by the presence of extra copies of the datA sequence alleviates replication problems in both the dnaX (Skovgaard & Lobner-Olesen, 2005) and the nrdA101 mutant (this work), whilst initiation defects caused by deletion of DnaA box R4 suppress replication elongation defects (Stepankiw et al, 2009).…”

“…Consistent with this, the presence of dnaA defective alleles, dnaA(Sx), suppresses the detrimental effect on DNA replication observed in mutants that have problems with the progression of forks due to the presence of defective subunits of DNA polymerase III coded by the dnaX gene (Gines-Candelaria et al, 1995;Blinkova et al, 2003;Skovgaard & Lobner-Olesen 2005). Furthermore, a lower availability of wild type DnaA protein induced by the presence of extra copies of the datA sequence alleviates replication problems in both the dnaX (Skovgaard & Lobner-Olesen, 2005) and the nrdA101 mutant (this work), whilst initiation defects caused by deletion of DnaA box R4 suppress replication elongation defects (Stepankiw et al, 2009). These observations, together with our data, are consistent with the idea that the progression of replication forks is not merely responsive to elongation factors (dNTP pools or proteins engaged in elongation) but also to the number of forks running along the chromosome.…”

“…The presence of dnaA defective alleles has been reported to reduce the time required for chromosomal elongation (C period) at permissive conditions and this effect might suppress the defects in replication of some DNA elongation mutants (Torheim et al, 2001;Skovgaard & Lobner-Olesen, 2005). We used the dnaA174 allele, which codes for a non-thermosensitive DnaA protein with a high ATPase activity, which in turn maintains a low DnaA-ATP level associated with a decreased the C period (Gon et al, 2006).…”

Chromosome replication status and DNA content at any cell age in a bacterial cell cycle

“…Determination of the rate of DNA synthesis at any time in an individual cell could lead to breakthroughs in our knowledge of how replication and the cell cycle are coregulated. This might shed some light, for instance, on the suggested reduction of the rate of DNA synthesis at a weakened replicating fork when a second replication round was initiated (Blinkova et al, 2003;Moreno et al, 2013;Salguero et al, 2011;Skovgaard and Løbner-Olesen, 2005). The algorithm should enable determination of the minimal replication rate under thymine limitation in which cell viability is maintained (Zaritsky et al, 2006) and a better description of nucleoid complexity during the cell cycle (Zaritsky, 2015).…”