Genetic instability and carcinogenesis

Keywords: Bloom syndrome, RecQ helicase, homologous recombination, replication stress, DNA damage response

Read the scientific activity report. (pdf 320Ko, last update 9, march 2009)

Fig. 1 

Bloom syndrome (BS) is a rare, human autosomal recessive disorder characterised by a strong predisposition to a wide range of cancers that commonly affect the general population. The hallmark of BS cells is a rate of sister chromatid exchange up to ten times higher than that found in normal cells, and this provides the only objective criterion for diagnosis of the disease (Fig. 1).

Bloom syndrome (BS) is caused by mutations in both copies of the BLM gene, which encodes BLM - a RecQ helicase with ATP-dependent 3'-5' DNA helicase activity. Several studies suggest that BLM is involved in restarting DNA replication when a replication fork stalls. The specific functions of the BLM protein, however, remain unknown. The key goals of our project are to characterise BLM's functions and to study the cellular and molecular consequences of the absence of a functional BLM protein. Thus, we hope to identify fundamental processes involved in maintaining genome integrity and preventing cancer.

Fig. 2 

In recent years, we have shown that BLM is regulated during the cell cycle: it accumulates to high levels in S phase, persists throughout G2 and M phase, and then disappears rapidly in G1 phase after cell division. We found that BLM participates in the cellular response to ionising radiation and to UVC radiation as well as to the inhibition of DNA synthesis by hydroxyurea. Consistent with this, BLM is involved in the repair of DNA double-strand breaks that can result from exposure of cells to ionising radiation or to hydroxyurea. On the basis of our findings, we propose a model for the role of BLM in DNA double-strand break repair (Fig. 2). In brief, we propose that BLM stays at the site of a DNA double-strand break while non-homologous DNA end joining (NHEJ) takes place. When NHEJ is activated, BLM is phosphorylated by DNA-PKcs, a key component of the NHEJ pathway, and this phosphorylation causes its dissociation from DNA. If NHEJ fails to repair the DNA break, then recombination takes place creating a single Holliday junction that is resolved by the reverse branch migration activity of BLM.

Fig. 3 

The BLM protein is active and hyperphosphorylated during mitosis. The introduction of a large number of DNA double-strand breaks into cells arrested in mitosis, however, results in BLM dephosphorylation and its recruitment into an insoluble protein fraction. Concomitant with this, the key mitotic kinase Cdc2 is inactivated. Since Cdc2 directly phosphorylates BLM in vitro, we propose that during mitosis BLM phosphorylation by Cdc2 leads to its exclusion from the chromatin and the nuclear scaffold, preventing its interference with mitotic processes such as chromosome condensation. This displaced BLM would form a pool of active protein that could be recruited rapidly to sites of repair if Cdc2 were subsequently inhibited in response to the presence of DNA damage or aberrant chromosome structures (Fig. 3).

We hypothesise that BLM-deficient cells survive by inducing a mechanism like the SOS response in bacteria (Amor-Guéret, 2006): an inducible system that allows bacteria to survive sudden increases in DNA damage. This response may operate during replication and/or during mitosis and may also occur in normal cells. This cellular escape process may therefore also be involved in the development of cancer in the general population.

We aim to test this hypothesis by investigating the role of BLM during mitosis and in the cellular response to replicative stress. We are also analysing the cellular and molecular consequences of the absence of a functional BLM protein during replicative stress, transcription, homologous recombination and mitosis.

Last update: July 2008

Key publications

2010

• Lahkim Bennani-Belhaj K, Rouzeau S, Buhagiar-Labarchède G, Chabosseau P, Onclercq-Delic R, Bayart E, Cordelières F, Couturier J and Amor-Guéret M.
  The Bloom syndrome protein limits the lethality associated with Rad51 deficiency
  Mol. Cancer Res., 8(3):385-394 - Abstract
  Little is known about the functional interaction between the Bloom's syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family.

2008

• Temime-Smaali N , Guittat L, Wenner T, Bayart E, Douarre C, Gomez D, Giraud-Panis MJ, Londono-Vallejo A, Gilson E, Amor-Guéret M & Riou JF.
  Topoisomerase III alpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres
  EMBO J., 27(10):1513-24 - Abstract
  Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.
  - Full version
• Amor-Guéret M, Dubois-d'Enghien C, Laugé A, Onclercq-Delic R, Barakat A, Chadli E, Bousfiha AA, Benjelloun M, Flori E, Doray B, Laugel V, Lourenço MT, Gonçalves R, Sousa S, Couturier J, & Stoppa-Lyonnet D.
  Three new BLM gene mutations associated with Bloom syndrome
  Genetic Testing, 12(2):257-261 - Abstract
  Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

2006

• Bayart E, Dutertre S, Jaulin C, Guo R-B, Xi X-G & Amor-Guéret M
  The Bloom syndrome helicase is a substrate of the mitotic cdc2 kinase
  Cell Cycle, 5, 1681-1686
• Amor-Guéret M
  Bloom syndrome, genomic instability and cancer: the SOS-like hypothesis
  Cancer Letters, 236, 1-12 - Abstract
  Bloom syndrome (BS) displays one of the strongest known correlations between chromosomal instability and an increased risk of malignancy at an early age. The prevention of genomic instability and cancer depends on a complex network of pathways induced in response to DNA damage and stalled replication forks, including cell-cycle checkpoints, DNA repair, and apoptosis. Several studies have demonstrated that BLM is involved in the cellular response to DNA damage and stalled replication forks. BLM interacts physically and functionally with several proteins involved in the maintenance of genome integrity and BLM is redistributed and/or phosphorylated in response to several genotoxic stresses. The data concerning the relationship between BLM and these cellular pathways are summarized and the role of BLM in the rescue of arrested replication forks is discussed. Moreover, I speculate that BLM deficiency is lethal, and that BLM-deficient cells escaping apoptotic death do so by constitutively inducing a bacterial SOS-like response including the induction of alternative replication pathway(s) dependent on recombination, contributing to the mutator and hyper-Rec phenotypes characteristic of BS cells. This mechanism may be dependent on the RAD51 gene family, and involved in carcinogenesis in the general population.

2004

• Bayart E, Grigorieva O, Leibovitch S, Onclercq-Delic R & M Amor-Guéret
  A major role for mitotic cdc2 kinase inactivation in the establishment of the mitotic DNA damage checkpoint
  Cancer Research, 64(24):8954-9 - Abstract
  Cdc2 kinase is inactivated when DNA damage occurs during the spindle assembly checkpoint. Here, we show that the level of mitotic Bloom syndrome protein phosphorylation reflects the level of cdc2 activity. A complete inactivation of cdc2 by either introduction of DNA double-strand breaks or roscovitine treatment prevents exit from mitosis. Thus, mitotic cdc2 inactivation plays a major role in the establishment of the mitotic DNA damage checkpoint. In response to mitotic cdc2 inactivation, the M/G(1) transition is delayed after releasing the drug block in nonmalignant cells, whereas tumor cells exit mitosis without dividing and rereplicate their DNA, which results in mitotic catastrophe. This opens the way for new chemotherapeutic strategies.

2003

• Onclercq-Delic R, Calsou P, Delteil C, Salles B, Papadopoulo D & M Amor-Guéret
  Possible anti-recombinogenic role of Bloom's syndrome helicase in double-strand break processing
  Nucleic Acids Res., 31, 6272-82 - Abstract
  Bloom's syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3'-5' DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.

2002

• Ababou M, Dumaire V, Lécluse Y & M Amor-Guéret.
  Bloom's syndrome protein response to ultraviolet C radiation and hydroxyurea mediated DNA synthesis inhibition
  Oncogene, 21, 2079-2088 - Abstract
  Bloom's syndrome (BS) arises through mutations in both copies of the BLM gene that encodes a RecQ 3'-5' DNA helicase. BS patients are predisposed to developing all the cancers that affect the general population, and BS cells exhibit marked genetic instability. We showed recently that BLM protein contributes to the cellular response to ionizing radiation by acting as downstream ATM kinase effector. We now show that following UVC treatment, BLM-deficient cells exhibit a reduction in the number of replicative cells, a partial escape from the G2/M cell cycle checkpoint, and have an altered p21 response. Surprisingly, we found that hydroxyurea-treated BLM-deficient cells exhibit an intact S phase arrest, proper recovery from the S phase arrest, and intact p53 and p21 responses. We also show that the level of BLM falls sharply in response to UVC radiation. This UVC-induced reduction in BLM does not require a functional ATM gene and does not result from a subcellular compartment change. Finally, we demonstrate that exposure to UVC and hydroxyurea treatment both induce BLM phosphorylation via an ATM-independent pathway. These results are discussed in the light of their potential physiological significance with regard to the role of BLM in the cellular pathways activated by UVC radiation or HU-mediated inhibition of DNA synthesis.