Cdc45 is a critical effector of myc-dependent DNA replication stress

Abstract

c-Myc oncogenic activity is thought to be mediated in part by its ability to generate DNA replication stress and subsequent genomic instability when deregulated. Previous studies have demonstrated a nontranscriptional role for c-Myc in regulating DNA replication. Here, we analyze the mechanisms by which c-Myc deregulation generates DNA replication stress. We find that overexpression of c-Myc alters the spatiotemporal program of replication initiation by increasing the density of early-replicating origins. We further show that c-Myc deregulation results in elevated replication-fork stalling or collapse and subsequent DNA damage. Notably, these phenotypes are independent of RNA transcription. Finally, we demonstrate that overexpression of Cdc45 recapitulates all c-Myc-induced replication and damage phenotypes and that Cdc45 and GINS function downstream of Myc.

Figure 1. Myc Triggers Premature Activation of Replication Origins

(A) Schematic representation of the experiments. (B) Types of DNA fibers scored in (C). Since digoxygenin-dUTP is not removed prior to addition of biotin-dUTP, DNA incorporating green and red nucleotides results in orange tracks. Scale bar is 10 kb. (C) Percentage of fibers that replicate early (green, before 60 min) or late (red, after 60 min) in control, Myc-, and mutant-Myc-supplemented extracts. Number of DNA molecules analyzed: N_Buffer = 120; N_Myc = 106. Error bars represent SD of the mean. See also Figure S1.

Figure 2. Myc Overexpression Decreases Interorigin Distance in Xenopus Extracts and Mammalian Cells

(A) Representative images of DNA molecules scored in (B) and (C). White arrows indicate the putative position of replication origins. Scale bar is 10 kb. (B) The interorigin distance is plotted against the percentage of DNA molecules in buffer and Myc-supplemented extracts. Number of origins analyzed: N_Buffer = 117 N_Myc = 118. (C) Number of origins was plotted against DNA length (kb). The numbers on/beside the circle represent the number of DNA fibers with a given number of origins. (D) B cells isolated from wild-type or λ-Myc mice were sequentially labeled with CldU and IdU. The combed DNA from cells was stained for early- (green) and late-(red) replicating tracts, and interorigin distances were plotted as described in (B). Error bars represent SD of the mean. See also Figure S1.

Figure 3. Myc Causes Replication Fork Stalling in Xenopus Extracts and Mammalian Cells

(A–D) The DNA fibers that had only one origin and contained forks that progressed either uni- or bidirectionally were analyzed. (A) Representative images of DNA molecules that incorporated digoxigenin (green) or biotin (red) in control (buffer) or upon Myc overexpression (Myc). Scale bar is 10 kb. (B) Fork progression ratio (FPR) of DNA fibers in control and Myc-supplemented extracts. FPR is calculated by measuring the lengths of red tracts and dividing the smaller value by the larger one. (C) Fraction of DNA fibers that had unidirectional fork progression in control and Myc-supplemented extracts. The dashed line for Myc represents the corrected value. n ≥ 100; the p value reflects the adjusted value. (D) B cells isolated from wild-type or λ-Myc mice were isolated and processed as in Figure 2D. Asymmetric forks were graphed as in Figure 3C. Error bars represent SD of the mean.

Figure 4. Cdc45 Recapitulates the Myc-Induced Replication Phenotype

(A) Schematic representation of the experiment. (B) Early- versus late-replicating DNA (green only versus red only) were plotted for control, Myc, Cdc45, and coexpression of Myc and Cdc45. (C) The interorigin distances for buffer, Myc, Cdc45, and coexpression of Myc and Cdc45 were measured and plotted as in Figures 1 and 2. (D) Graph showing the fraction of DNA that had unidirectional fork progression (FPR < 0.25) in control, Myc-, Cdc45-, and Cdc45- and Myc-supplemented extracts; n ≥ 100. (E) Interorigin distance was measured after addition of recombinant GINS protein as described in Figure 2. (F) Asymmetric forks were measured after addition of recombinant GINS as described in Figure 3. Error bars represent SD of the mean. See also Figure S2.

Figure 5. Cdc45 and GINS Are Required for Myc-Dependent Phenotypes

(A) Early- versus late-replicating DNA fibers (green only versus red only) for buffer or Myc-supplemented extracts in mock- or Cdc45-depleted extracts. (B) Interorigin distances for buffer (control) or Myc extracts in mock- or Cdc45-depleted conditions. (C) Asymmetric fork progression for buffer or Myc extracts in mock- or Cdc45-depleted conditions were plotted as described in Figure 2; n ≥ 100. (D and E) The GINS complex was partially depleted using Psf2 antibody. The effect of overexpressing Myc in mock- or GINS-depleted extracts was evaluated. The experimental setup and measurement of interorigin distances (D) and asymmetric forks (E) are described earlier (Figures 2 and 3). Error bars represent SD of the mean. See also Figure S3.

Figure 6. Mechanisms of Myc-Dependent DNA Damage

(A) Chromatin was incubated with extracts that were supplemented with buffer, Myc, or Cdc45 protein for 45 min. Chromatin-bound proteins were isolated and immunoblotted with phospho-ATM (pATM) and γH2AX antibodies (H2AX). Histone H3 was used as loading control. The numerical values indicate densitometric analysis of band intensity using ImageJ. (B) Mock- or Cdc45-depleted extracts were supplemented with sperm chromatin and incubated for 1 hr. Chromatin-bound pATM and γH2AX were assessed as in (A). Band intensities were quantified using ImageJ; control/buffer was assigned a value of 1. Depleted extracts were compared with their corresponding mock-depleted controls. (C) Sperm chromatin was replicated in extracts supplemented with P³² dCTP and increasing amounts of recombinant p27 protein. The replication assay was performed as described in Experimental Procedures. Radioactivity incorporated into DNA was quantified and plotted. The arrow indicates the concentration of p27 that inhibited DNA replication by 66%. (D) Chromatin was isolated from extracts supplemented with buffer (lane 1), Myc alone (lane 2), p27 alone (lane 3), or Myc and p27 (lane 4). The concentration of p27 protein that inhibits DNA replication by 66% (as determined from Figure 6C) was used. Chromatin-bound proteins were blotted with Cdc45 antibodies. Histone H3 was used as a loading control.