• Blogs
  • Leo Bertram
  • Take A Look The following And Understand The Best Way To Master SAR245409 Quickly

Take A Look The following And Understand The Best Way To Master SAR245409 Quickly

?coli cells and discovered that acute amino acid starvation not only inhibited replication initiation, but also modestly reduced the rate of replication elongation. We found that (p)ppGpp was both

necessary and sufficient to inhibit replication elongation independently of its effect on transcription. We further observed GSK2126458 nmr that (p)ppGpp inhibited replication elongation quantitatively in both E.?coli and B.?subtilis, with higher concentrations of (p)ppGpp inhibiting replication elongation more strongly. This modulation may be the manifestation of a conserved regulatory mechanism in bacteria to maintain genome integrity. We investigated whether replication elongation is affected by amino acid starvation in living E.?coli cells. We monitored replication fork progression in a synchronized population of E.?coli cells using genomic microarrays (Khodursky et?al., 2000; Tehranchi et?al., 2010). Cells carrying

a temperature sensitive (dnaC2) mutant of the helicase loader DnaC (Carl, 1970) were grown in minimal medium; replication was synchronized by shifting the culture to 42��C to inhibit replication initiation while allowing the current round of replication to complete, and then returned to 30��C to allow synchronized release of a new round of replication (Fig.?1A). Serine hydroxamate (SHX), which mimics amino acid starvation and induces (p)ppGpp to high levels (Pizer and Merlie, 1973), was added 10?min after temperature downshift and the gene dosage profiles at 10?min (Fig.?1B), 35?min (Fig.?1C) and 45?min (Fig.?1D) after temperature downshift were obtained. Replication fork positions were calculated as the midpoint of gene

dosage between replicated and unreplicated regions (Fig.?1E). The rates of replication elongation, calculated by linear regression of replication fork positions at 10, 35 and 45?min (Table?1), were found to be reduced by 13?��?2% upon starvation, illuminating a modest but significant (P?<?0.01, Mann�CWhitney U-test), previously unknown effect of amino acid starvation on the replication elongation rate in E.?coli. Inhibition of replication elongation in wild-type cells may be due to amino acid starvation alone or due to the accumulation of (p)ppGpp upon starvation. To determine whether (p)ppGpp induction is necessary for this inhibition, we examined cells lacking the (p)ppGpp synthetase RelA that is responsible for production of (p)ppGpp in response to amino acid starvation (Potrykus and Cashel, 2008). Replication fork progression was no longer affected by starvation in dnaC2 ��relA cells (Fig.?1F), indicating that inhibition of replication elongation requires (p)ppGpp induction in E.?coli. As (p)ppGpp induction upon starvation in E.?coli cells results in a modest reduction of replication elongation rate, we examined whether further increasing (p)ppGpp concentration inhibits replication elongation more strongly.</p>