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. 2014 Nov 26;10(10):1193-202.
doi: 10.7150/ijbs.11066. eCollection 2014.

SIRT1 deacetylates TopBP1 and modulates intra-S-phase checkpoint and DNA replication origin firing

Rui-Hong Wang  1 , Tyler J Lahusen  2 , Qiang Chen  2 , Xiaoling Xu  1 , Lisa M Miller Jenkins  3 , Elisabetta Leo  4 , Haiqing Fu  4 , Mirit Aladjem  4 , Yves Pommier  4 , Ettore Appella  3 , Chu-Xia Deng  1
Affiliations

SIRT1 deacetylates TopBP1 and modulates intra-S-phase checkpoint and DNA replication origin firing

Rui-Hong Wang et al. Int J Biol Sci. .

Abstract

SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.

Keywords: DNA replication fork; Genetic stability.; Intra-S-phase checkpoint; SIRT1; TopBP1.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Loss of SIRT1 impairs intra-S-phase checkpoint. (A, B) Percentage of BrdU incorporation of primary SIRT1-WT and SIRT1-MT MEF cells under regular culture condition (A) and upon HU treatment (B). (C) Number of MEF cells at passage 1-3. (D) Dose response of DNA synthesis 30 min post γ-irradiation, as detected by thymidine incorporation assay. (E) Time course response of DNA synthesis after 10 Gy γ-irradiation. Three pairs of primary MEF cells at passage 1 or 2 were used for this assay. (F) Response of immortalized SIRT1-WT or MT MEFs upon CPT treatment. Cells were exposed to 5 μM CPT for 2 and 4 hours, and DNA synthesis was assessed after 30 minutes of BrdU labeling by FACs analysis. * P<0.05 and ** P<0.01: comparison between SIRT1-MT and SIRT1-WT cells.
Figure 2
Figure 2
SIRT1 modulates velocity and inter-origin distance of DNA replication. (A, B) DNA combing analysis of replication fork velocity (A) and inter-origin distance (B) in SIRT1-WT and SIRT1-MT MEFs with or without CPT treatment. (C-F) Scatter plots showing replication speed (Kb/min) of left and right arms of replication forks in SIRT1-WT and SIRT1-MT MEFs without (C, D) and with (E, F) 5 μM CPT for one hour.
Figure 3
Figure 3
SIRT1 interacts with proteins involved in the regulation of DNA replication. (A, B) Interaction between ectopically over-expressed Flag-SIRT1 with TopBP1 (A) and CDC7 (B) as detected by immuno-precipitation. (C-E) Reciprocal immuno-precipitation with endogenous proteins to confirm interaction between SIRT1 versus TopBP1,CHK1 and PARP1 (C); TopBP1 versus SIRT1 (D); and CHK1 versus SIRT1 (E). (F) DNA replication stress enhances interaction of SIRT1 with TopBP1 and CHK1. 293T cells were treated with different doses of HU for 4 hours. Then SIRT1 was immuno-precipitated and TopBP1 and CHK1 were detected by western blot.
Figure 4
Figure 4
SIRT1 deacetylates TopBP1 to affect DNA replication. (A). Acetylation of TopBP1 is regulated by SIRT1 activity. 293T cells were transfected with a Flag-TopBP1 plasmid in combination with GFP, SIRT1 (T1), SIRT1-HY (T1-HY) or shSIRT1 (shT1), respectively. TopBP1 was pulled down with anti-Flag antibody, and then the immuno complexes were western blotted with an antibody against acetyl-lysine. (B) Potential acetylation sites in TopBP1 as revealed by in-silico analysis in combination with mass spectrometry analysis. (C) MS/MS spectra for an acetylated peptide of TopBP1 containing acetylation of Lys475. (D) Effect of SIRT1 and SIRT1-HY on acetylation of TopBP1. (E) Effect of ectopic expression of TopBP1 constructs on DNA replication as determined by BrdU incorporation in primary SIRT1-MT and SIRT1-WT MEF cells after release from HU treatment. ## P<0.01: comparison between SIRT1-WT and MT cells with the same transfection condition (i.e. control vector vs control vector, and 3R vs 3R). * P<0.05 and ** P<0.01: comparison between control with other transfection conditions (i.e. control vs 3R, and control vs 3Q).
Figure 5
Figure 5
SIRT1 deficiency affects DNA replication and is critical for genome stability. (A, B) DNA replication analysis by IdU-CIdU labeling in SIRT1-WT (A) and SIRT1-MT (B) MEFs, as indicated on the top of the panel. Briefly, cells were labeled with IdU for 20 min followed by 10 mM HU treatment for 4 hours. After removing HU, cells were labeled with CIdU for 60 min before processing for imaging. Three pairs of MEF cells at passage 2 were utilized. (C) Chromosome spreads showing abnormal chromosome structures (arrows) in mutant but not in WT cells. (D) Percentage of abnormal chromosomes.
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