Nutritional control of elongation of DNA replication by (p)ppGpp

Abstract

DNA replication is highly regulated in most organisms. Although much research has focused on mechanisms that regulate initiation of replication, mechanisms that regulate elongation of replication are less well understood. We characterized a mechanism that regulates replication elongation in the bacterium Bacillus subtilis. Replication elongation was inhibited within minutes after amino acid starvation, regardless of where the replication forks were located on the chromosome. We found that small nucleotides ppGpp and pppGpp, which are induced upon starvation, appeared to inhibit replication directly by inhibiting primase, an essential component of the replication machinery. The replication forks arrested with (p)ppGpp did not recruit the recombination protein RecA, indicating that the forks are not disrupted. (p)ppGpp appear to be part of a surveillance mechanism that links nutrient availability to replication by rapidly inhibiting replication in starved cells, thereby preventing replication-fork disruption. This control may be important for cells to maintain genomic integrity.

Fig. 1. Monitoring progression and arrest of replication forks

(A) Diagram of the circular B. subtilis chromosome. The origin (oriC) and terminus (ter) of replication, directions of fork progression (gray arrows), and regions (light gray bars) proposed to contain specific sites for replication arrest (Autret et al., 1999; Levine et al., 1995; Levine et al., 1991) are indicated. (B–F) Replication in the dnaBts mutant (KPL69) was monitored using DNA microarrays to measure gene dosage throughout the chromosome. Relative DNA levels (log₂) were determined by co-hybridization of replicating and pre-initiation reference DNA to whole-genome microarrays and plotted as a function of gene position. oriC is in the middle (0 Mbp), and ter is to the left (~−2.2 Mbp) and right (~2.0 Mbp). Arrows indicate the positions of the replication forks, defined as the boundaries between the replicated and un-replicated genes. Each panel shows data from one experiment and is representative of multiple experiments. Samples were taken immediately before (0 min) (B), and 20 (C), 40 (D), and 80 min (E) after initiation of replication. (F) RHX was added at the time of initiation of replication and cells were sampled 40 min later. (G, H) Distances of the left (empty symbols, dashed lines) and right (filled symbols, solid lines) replication forks from oriC are plotted as a function of time after initiation of replication. Cells were synchronized for the replication cycle and DNA content was determined as above. Where indicated, RHX, SHX, or norvaline was added 20 min (vertical arrows) after replication initiation. (G) Cells without (squares) and with RHX (triangles). The location of the proposed replication arrest sites (Autret et al., 1999; Levine et al., 1995; Levine et al., 1991) is indicated (gray bar) on the y-axis. (H) Cells without (squares) and with SHX (triangles) or norvaline (circles)

Fig. 2. Replication arrest induced by amino acid starvation depends on relA but not rtp

(A–F) Cells were synchronized for the replication cycle and DNA content analyzed as in Fig. 1. RHX was added 20 min after replication initiation. Samples were taken 20 min after replication initiation, immediately before addition of RHX (panels A, C, E) and 60 min after replication initiation (40 min after addition of RHX) (panels B, D, F). Arrows indicate positions of replication forks. (A, B) rtp⁺ relA⁺ cells (KPL151); (C, D) Δrtp (JDW163); (E, F) ΔrelA (JDW184). (G, H) Effects of amino acid starvation on asynchronous replication. Cells were grown to mid-exponential phase (OD₆₀₀~0.3) at 37°C. The relative rate of DNA synthesis (incorporation of ³H-thy into DNA of treated samples normalized to untreated) is plotted as a function of time after amino acid starvation. (G) Relative rate of DNA synthesis after treatment with RHX. filled squares, relA⁺ rtp⁺ (JH642); open triangles, Δrtp (JDW119); open circles, ΔrelA (BB914). (H) Relative rate of DNA synthesis in wild type (JH642) after treatment with SHX (filled squares) or norvaline (open squares).

Fig. 3. Starvation-induced replication arrest is dependent on accumulation of (p)ppGpp and independent of transcription

Wild type cells (JH642) were grown at 37°C and treated as indicated. open diamonds, rifampicin was added at t=0. filled squares, RHX was added at t=0. open squares and dotted lines, rifampicin was added at t=0 and RHX was added 1.5 min later. filled triangles, decoyinine was added at t=0. filled diamonds, untreated. (A) Rifampicin rapidly inhibits transcription. The rate of transcription was measured by pulse labeling cells with ³H-uridine for 1 min, measuring incorporation of ³H-uridine into TCA-precipitable fractions, and normalizing to an untreated sample. The normalized rate is plotted as a function of time after addition of rifampicin. (B) Rates of replication. The rate of replication was measured as described (Fig 2G, H) and is plotted as a function of time after the indicated treatment. (C) Addition of rifampicin increases uptake of ³H-thy into cells. The rate of uptake was obtained by pulse labeling cells with ³H-thy for 2 min, measuring ³H-thy retained in cells, normalizing to cells at t=0, and plotting the normalized rate as a function of time after indicated treatment. (D, E, F) Effects of indicated treatments on levels of ppGpp (D), GTP (E), and ATP (F). Cultures were labeled with ³²P-inorganic phosphate and nucleotides were extracted, separated by TLC, and quantified using a PhosphorImager. The total phosporimager count for each spot is plotted as a function of time after the indicated treatment. Results from one experiment are shown and are representative of ≥2 experiments. Not shown: pppGpp accumulated with similar kinetics as ppGpp, but pppGpp levels were ~4-fold higher than those of ppGpp.

Fig. 4. Effects of (p)ppGpp and amino acid starvation on DNA polymerase and primase activity

(A, B) Activity of purified B. subtilis DNA polymerase PolC (A) or primase DnaG (B) in vitro. PolC gap filling activity (A) or primase activity (B) is plotted as a function of indicated concentrations of GDP (open triangles), ppGpp (filled squares) and pppGpp (open circles). Primase activity in the presence of 25 μg/ml of rifampicin is shown. Primase activity in the absence of rifampicin is similar (data not shown). (C) Increase in plasmid ssDNA upon treatment with RHX. DNA was purified from cells containing plasmid pHV1610-1 (JDW280). Plasmid ssDNA and dsDNA forms were separated on agarose gels, transferred to nylon membranes, and hybridized with a labeled plasmid probe. The percentage of ssDNA relative to the covalently closed circular dsDNA of pHV1610-1 was determined for cells untreated or treated with RHX for 20 min. Inhibition of primase is known to cause an ~2-fold increase in pHV1610-1 ssDNA (Bruand et al., 1995).

Fig. 5. Arrest of replication by amino acid starvation does not induce formation of RecA-GFP foci. Focus formation was monitored by fluorescence microscopy. Cells with a functional recA-gfp fusion (LAS40) were grown at 30°C and treated as indicated for 40 min before observation

(A–D) Micrographs of cells containing RecA-GFP (green diffuse background or foci) with membranes stained with FM4-64 (red). Cells were untreated (A); treated with HPUra (38 μg/ml) (B) which binds to and inhibits DNA polymerase; treated with hydroxyurea (4 mg/ml) (C) which depletes dNTP pools; or treated with RHX (D). (E) Fractions of cells with discernable RecA-GFP foci and the treatments and total number of cells measured (n) are indicated.