DNA replication is regulated in response to environmental constraints such as

DNA replication is regulated in response to environmental constraints such as nutrient availability. activity (Wang et al. 2007 Paradoxically primase activity is also directly inhibited by (p)ppGpp (Maciag et al. 2010 Rymer et al. 2012 although decades of classical and modern experiments have not revealed any inhibitory effect of amino acid starvation on replication elongation (Lark and Lark 1966 Billen and Hewitt 1966 Marsh and Hepburn 1980 Levine et al. 1991 Ferullo and Lovett 2008 Tehranchi et al. 2010 This disparity between and results suggests that either (p)ppGpp also inhibits replication elongation in or is conserved in divergent bacteria we quantified genome-wide replication fork progression in 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 that (p)ppGpp inhibited replication elongation quantitatively in both and cells. We monitored replication fork progression in a synchronized population of cells using genomic microarrays (Khodursky et al. 2000 Tehranchi et al. 2010 Cells carrying a temperature sensitive (Δcells (Figure 1F) indicating that inhibition of replication elongation requires (p)ppGpp induction in cells results in CASIN a modest reduction of replication elongation rate we examined whether further increasing (p)ppGpp concentration inhibits replication elongation more strongly. This can be achieved by deleting (Somerville and Ahmed 1979 (Figure 2A). Using Thin Layer Chromatography (TLC) we confirmed that in Δcells ppGpp was induced by SHX to similar levels as in wild-type cells but pppGpp levels were ~2 fold higher (Figure 2B-D; Table S1). Levels of GTP a precursor of pppGpp and a substrate for primase were reduced similarly in the presence or absence of GppA (Figure 2E; Table S1). Figure 2 Deletion of Results in Higher pppGpp Levels upon Amino Acid Starvation in (Figure 3A-D). While replication elongation rates were not significantly reduced by deletion in untreated cells upon starvation elongation rates were reduced by 35±3% (p < 0.01 Mann-Whitney U test) a 2-3 fold further reduction compared with starved cells (13±2%). Figure 3 Replication Elongation Rates are More Strongly Reduced in Amino Acid-Starved ΔCells To rule CASIN out the possibility that this reduction stems from a synthetic effect between the deletion and the allele we measured CASIN replication rates in Δcells with wild-type by monitoring the incorporation of 3H-thymidine into DNA. While inhibition IMPG1 antibody of replication initiation results in a gradual decrease of 3H-thymidine incorporation over the course of a replication cycle inhibition of elongation results in a rapid decrease of 3H-thymidine incorporation. We observed a rapid decrease in the rate of 3H-thymidine incorporation during amino acid starvation in wild-type cells and found that deletion resulted in a significant further reduction (Figure 3E). It has been shown that (p)ppGpp induction also decreased the uptake of thymidine (Lin-Chao and Bremer 1986 which contributed to the decrease in 3H-thymidine incorporation. However deletion of CASIN did not further decrease thymidine uptake (Figure 3E) suggesting that the difference in thymidine incorporation we observed in SHX-treated Δcells was CASIN due to reduction of DNA replication elongation rates in the presence of wild-type deletion in cells devoid of any (p)ppGpp via removal of both (p)ppGpp synthetases: RelA and SpoT. There was no appreciable reduction of the replication rate in ΔΔΔcells upon SHX treatment (Figure 3F) confirming that the inhibition of replication elongation in the Δmutant resulted from increased pppGpp levels. (p)ppGpp is Sufficient to Slow Replication Elongation in E. coli In (Figure 1-3) and (Wang et al. 2007 the inhibitory effects of (p)ppGpp on replication elongation were revealed only under amino acid starvation. Consequently we wanted to determine whether (p)ppGpp induction only not in combination with amino acid starvation was adequate to reduce replication elongation rates in deletion (Number 4A-C; Table S1). RelA* induction reduced GTP levels to the same extent.