Chk1 promotes replication fork progression by controlling replication initiation

Abstract

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity.

Fig. 1.

Effect of the Cdk inhibitor roscovitine on origin firing and replication fork speeds. (A) Labeling protocols for DNA fiber analysis. U2OS cells were pretreated with 25 μM roscovitine (rosc) or an equal volume of DMSO (control) for 1 hour and then pulse labeled with CldU and IdU for 20 minutes each in presence of rosc or DMSO. CldU was detected using a specific primary antibody and a secondary antibody in red. IdU was detected using specific primary antibody and a secondary antibody in green. (B) Representative images of replication tracks from cells treated with DMSO or rosc. (C) Quantification of origin firing in cells treated with DMSO or rosc. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. (D) Distribution of replication fork speeds in cells treated as in C. (E) Average replication fork speeds in cells treated with DMSO or rosc. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t-test, ** p < 0.01).

Fig. 2.

Cotreatment with roscovitine partially rescues fork slowing Chk1 inhibitor-treated cells. (A) Representative images of replication tracks from cells treated with DMSO, CEP-3891 (CEP), or CEP + roscovitine (rosc). (B) Quantification of origin firing in cells treated with DMSO, CEP, or CEP + rosc. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. (C) Distribution of replication fork speeds in cells treated as in B. (D) Average replication fork speeds in cells treated with DMSO, CEP, rosc, or CEP + rosc. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t-test, * p < 0.05, ** p < 0.01).

Fig. 3.

Chk1 inhibited cells display normal S phase length while Cdk inhibition slows S phase progression. (A) Flow cytometry profiles of progression through S phase in presence of DMSO, CEP-3891, or roscovitine. Asynchronously growing U2OS cells were pulse labeled with IdU for 20 min and released into medium containing drug or DMSO. Nuclei were immunostained for IdU and stained for DNA using propidium iodide (PI). Profiles show PI distribution of cells stained for IdU (which were in S phase during pulse label) after 0, 3, 6, or 9 hours (see also Fig. S4). (B) IdU-labeled cells in mid-S phase after 3, 6, or 9 hours release from IdU as percentage of fraction at 0 hours. Means and standard deviation (S.D.) (bars) of three independent experiments are shown.

Fig. 4.

Cdc7 codepletion rescues fork slowing in Chk1-depleted cells. (A) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control siRNA. (B) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. (C) Distribution of replication fork speeds in Cdc7- or control-depleted cells. (D) Distribution of replication fork speeds in Chk1- or control-depleted cells. (E) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. (F) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t-test, * p < 0.05).