Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Feb 17;29(4):806-18.
doi: 10.1038/emboj.2009.385. Epub 2010 Jan 7.

ATR activation and replication fork restart are defective in FANCM-deficient cells

Rebekka A Schwab  1 , Andrew N Blackford, Wojciech Niedzwiedz
Affiliations

ATR activation and replication fork restart are defective in FANCM-deficient cells

Rebekka A Schwab et al. EMBO J. .

Abstract

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA-ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR-mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
FANCM-deficient cells are hypersensitive to agents interfering with replication, display altered global DNA replication, and accumulate spontaneous DNA damage. (A) Cellular sensitivity of WT and ΔFANCM cells to camptothecin (CPT), hydroxyurea (HU), and aphidicolin (APH) as measured by MTS survival assay. Mean values of three independent experiments are shown ±s.e.m. (B) Representative images of actual fibres from WT and ΔFANCM cells. (C) Replication fork velocity in the WT DT40 and ΔFANCM cells used in this study. A minimum of 200 tracts were measured per experiment. Mean values for three independent experiments are shown ±s.e.m. (D) Five classes of replication structures and (E) the relative frequency of occurrence of these different classes. A minimum of 200 fibres were scored per experiment. Mean values of three independent experiments are shown ±s.e.m. (F) γ-H2AX foci in untreated WT and ΔFANCM cells. Left panel shows representative images of γ-H2AX foci. Right panel shows quantification of percentage cells exhibiting γ-H2AX foci out of more than 200 cells per experiment. Bars represent mean values from three independent experiments ±s.e.m.
Figure 2
Figure 2
Analysis of replication fork dynamics and restart in CPT-treated WT and ΔFANCM cells. (A) Top—schematic of the fibre labelling experiment, below—replication fork rates shown as frequency distribution in WT DT40 and ΔFANCM cells treated with CPT for 20 min during the second labelling period. The x axis represents the ratio between the tract length of the first label (IdU) divided by the tract length of the second label (CldU). (B) Top—schematic of the fibre labelling experiment for analysis of replication fork recovery and representative images of actual fibres from WT and ΔFANCM cells, below—effect of 90 min exposure to CPT on replication fork activity in the absence (C) and presence of roscovitine (D). The bars represent mean values from three independent experiments ±s.e.m.
Figure 3
Figure 3
Recovery of DNA synthesis and suppression of excess origin firing in ΔFANCM cells is caffeine sensitive but ATM- and Chk1 independent. (A) Western blot showing levels of Chk1 phosphorylation using a phospho-specific antibody to phosphorylated serine 345 (Chk1-S345) on extracts from WT DT40 and ΔFANCM cells treated with CPT for the indicated periods of time. Histone H3 was used as a loading control. Analysis of recovery of DNA synthesis and activation of latent origins in WT DT40 and ΔFANCM cells treated for 90 min with CPT in the presence and absence of indicated inhibitors (B) UCN-01; (C) caffeine and (D) ATM inhibitor. The bars represent mean values from three independent experiments ±s.e.m.
Figure 4
Figure 4
FANCM is required for efficient activation of ATR in response to CPT by promoting TopBP1 loading on chromatin. (A) Western blot showing phosphorylation status of SMC1 in whole cell extracts from WT and ΔFANCM DT40 cells treated for the indicated times with 1 μM CPT. Phosphorylated SMC1 was detected using a phospho-specific antibody raised against human SMC1 phosphorylated at serine 966 (SMC1-S966). Total SMC1 was used as a loading control. (B) Western blot showing RPA and ATR loading onto chromatin in response to 1 μM CPT treatment in WT and ΔFANCM DT40 cells. Histone H3 was used as a loading control. (C) Western blot for chromatin loading of TopBP1 in WT, ΔFANCM, and FANCM K-IN (knockin) cells after treatment with CPT with SMC1 as the loading control.
Figure 5
Figure 5
ATR and PLK kinases control recovery from replicative stress in ΔFANCM cells. (A) Western blot showing MCM2 levels in chromatin fractions derived from WT DT40 and ΔFANCM cells treated with CPT for the indicated periods of time. The arrows indicate the band shift resulting from MCM2 phosphorylation after CPT treatment. Histone H3 was used as a loading control. (B) MCM2 immunoprecipitated from WT and ΔFANCM was blotted for phosphorylation at serine 108 (pMCM2-S108) in cells treated with or without CPT and caffeine. (C) Effect of PLK1 inhibitors on recovery of DNA synthesis and activation of latent origins in WT DT40 and ΔFANCM cells treated for 90 min with CPT in the presence and absence of indicated inhibitors. The bars represent mean values from three independent experiments ±s.e.m.
Figure 6
Figure 6
FANCM promotes replication fork recovery and inhibition of latent origins by a mechanism independent from its role in the FA pathway. Analysis of recovery of DNA synthesis and activation of latent origins in WT, ΔFANCM, ΔFANCC, and ΔFANCI DT40 cells treated with CPT for 90 min. The bars represent mean values from three independent experiments ±s.e.m.
See this image and copyright information in PMC

Comment in

  • FANCM: fork pause, rewind and play.
    Collis SJ, Boulton SJ. Collis SJ, et al. EMBO J. 2010 Feb 17;29(4):703-5. doi: 10.1038/emboj.2009.415. EMBO J. 2010. PMID: 20160754 Free PMC article.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abraham RT (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 15: 2177–2196 - PubMed
    1. Bakker ST, van de Vrugt HJ, Rooimans MA, Oostra AB, Steltenpool J, Delzenne-Goette E, van der Wal A, van der Valk M, Joenje H, Te Riele H, de Winter JP (2009) Fancm-deficient mice reveal unique features of Fanconi anemia complementation group M. Hum Mol Genet 18: 3484–3495 - PubMed
    1. Ciccia A, Ling C, Coulthard R, Yan Z, Xue Y, Meetei AR, Laghmani el H, Joenje H, McDonald N, de Winter JP, Wang W, West SC (2007) Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM. Mol Cell 25: 331–343 - PubMed
    1. Cimprich KA, Cortez D (2008) ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol 9: 616–627 - PMC - PubMed
    1. Collis SJ, Barber LJ, Clark AJ, Martin JS, Ward JD, Boulton SJ (2007) HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability. Nat Cell Biol 9: 391–401 - PubMed

Publication types

MeSH terms