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Bloom's syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest

Blooms syndrome (BS) is a rare genetic disorder characterized by a broad range of symptoms and, most importantly, a predisposition to many types of cancers. Cells derived from patients with BS exhibit an elevated rate of somatic recombination and hypermutability, supporting a role for bleomycin (BLM) in the maintenance of genomic integrity. BLM is thought to participate in several DNA transactions, the failure of which could give raise to genomic instability, and to interact with many proteins involved in replication, recombination, and repair. In this study, we show that BLM function is specifically required to properly relocalize the RAD50/MRE11/NBS1 (RMN) complex at sites of replication arrest, but is not essential in the activation of BRCA1 either after stalled replication forks or γ-rays. We also provide evidence that BLM is phosphorylated after replication arrest in an Ataxia and RAD3-related protein (ATR)-dependent manner and that phosphorylation is not required for subnuclear relocalization. Therefore, in ATR dominant negative mutant cells, the assembly of the RMN complex in nuclear foci after replication blockage is almost completely abolished. Together, these results suggest a relationship between BLM, ATR, and the RMN complex in the response to replication arrest, proposing a role for BLM protein and RMN complex in the resolution of stalled replication forks.

Werner's syndrome protein is phosphorylated in an ATR/ATM-dependent manner following replication arrest and DNA damage induced during the S phase of the cell cycle.

Werners syndrome (WS) is an autosomal recessive disorder, characterized at the cellular level by genomic instability in the form of variegated translocation mosaicism and extensive deletions. Individuals with WS prematurely develop multiple age-related pathologies and exhibit increased incidence of cancer. WRN, the gene defective in WS, encodes a 160-kDa protein (WRN), which has 3′–5′exonuclease, DNA helicase and DNA-dependent ATPase activities. WRN-defective cells are hypersensitive to certain genotoxic agents that cause replication arrest and/or double-strand breaks at the replication fork, suggesting a pivotal role for WRN in the protection of the integrity of the genoma during the DNA replication process. Here, we show that WRN is phosphorylated through an ATR/ATM dependent pathway in response to replication blockage. However, we provide evidence that WRN phosphorylation is not essential for its subnuclear relocalization after replication arrest. Finally, we show that WRN and ATR colocalize after replication fork arrest, suggesting that WRN and the ATR kinase collaborate to prevent genome instability during the S phase.

Werner syndrome helicase activity is essential in maintaining fragile site stability

WRN is a member of the RecQ family of DNA helicases implicated in the resolution of DNA structures leading to the stall of replication forks. Fragile sites have been proposed to be DNA regions particularly sensitive to replicative stress. Here, we establish that WRN is a key regulator of fragile site stability. We demonstrate that in response to mild doses of aphidicolin, WRN is efficiently relocalized in nuclear foci in replicating cells and that WRN deficiency is associated with accumulation of gaps and breaks at common fragile sites even under unperturbed conditions. By expressing WRN isoforms impaired in either helicase or exonuclease activity in defective cells, we identified WRN helicase activity as the function required for maintaining the stability of fragile sites. Finally, we find that WRN stabilizes fragile sites acting in a common pathway with the ataxia telangiectasia and Rad3 related replication checkpoint. These findings provide the first evidence of a crucial role for a helicase in protecting cells against chromosome breakage at normally occurring replication fork stalling sites.

Replication fork stalling in WRN-deficient cells is overcome by prompt activation of a MUS81-dependent pathway

Failure to stabilize and properly process stalled replication forks results in chromosome instability, which is a hallmark of cancer cells and several human genetic conditions that are characterized by cancer predisposition. Loss of WRN, a RecQ-like enzyme mutated in the cancer-prone disease Werner syndrome (WS), leads to rapid accumulation of double-strand breaks (DSBs) and proliferating cell nuclear antigen removal from chromatin upon DNA replication arrest. Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling. Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest. Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phase

In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2+/+ and MSH2−/− mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells. Finally, MSH2−/− cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.

Werner’s syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage

Werners syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Werner syndrome protein and the MRE11 complex are involved in a common pathway of replication fork recovery.

Werner syndrome (WS) is an autosomal recessive disease that predisposes individuals toa wide range of cancers. The gene mutated in WS, WRN, encodes a member of the RecQfamily of DNA helicases. The precise DNA metabolic processes in which WRN participatesremain to be elucidated. However, it has been proposed that WRN might play an importantrole in the maintenance of genetic stability during DNA replication, possibly cooperatingwith other proteins. Here, we show that, following DNA replication arrest, WRN associatesand colocalises with the MRE11 complex at PCNA sites. We also provide evidence thatboth WRN/MRE11 complex association and proper WRN relocalisation after HU treatmentrequire a functional MRE11 complex. We demonstrate that mutations altering thefunctionality of WRN or that of the MRE11 complex result in chromosomal breakage duringDNA replication and enhanced cell death following replication arrest. Finally, we show thatthe DNA breakage in replicating cells and apoptosis observed in WS are not enhanced byconcomitant knock down of MRE11 by RNAi, indicating that WRN and MRE11 complexact in a common pathway. These results suggest a functional relationship between WRNand the MRE11 complex in response to replication fork arrest, disclosing a common actionof WRN and the MRE11 complex in the pathway(s) preserving genome stability duringDNA replication.

Fanconi Anemia Proteins and the S Phase Checkpoint

DNA interstrand crosslinks (ICLs) repair represents a formidable task for mammalian cells. Indeed, such DNA lesions, bridging both opposite DNA helices, function as a roadblock for every DNA transaction, in particular DNA replication. The eight Fanconi anemia (FA) proteins interact in a common pathway that is thought to be central in ICLs sensing/repair. Interestingly, FA cells, either mutated in one of the proteins composing the FA core complex or in the downstream FA protein FANCD2, exhibited a partial intra-S checkpoint defect in response to crosslinked DNA. Most importantly, the FA proteins work in the ATR-NBS1 branch of the ICL-induced checkpoint pathway as demonstrated by knocking-down CHK1 or MRE11 expression in a FA background. Even though our data disclose a clear functional role for the FA proteins in the intra-S checkpoint response it does not give a definite answer on what FA proteins do in this process and how they participate in the suppression/restart of DNA synthesis.It seems conceivable that FA proteins participate in the process involved in the recovery of stalled replication forks, a common event in proliferating cells, possibly ensuring correct replication fork repair by homologous recombination.

Transcription coupled repair efficiency determines the cell cycle progression and apoptosis after UV exposure in hamster cells

Nucleotide excision repair (NER) is a major pathway for the removal of bulky adducts and helix distorting lesions from the genomic DNA. NER is highly heterogeneous across the genome and operates principally at different levels of hierarchy. Transcription coupled repair (TCR), a special sub-pathway of NER and base excision repair (BER), is critical for cellular resistance after UV irradiation in mammalian cells. In this study, we have investigated the effects of UV-C irradiation on cell cycle progression and apoptosis in G1 synchronised isogenic hamster cell lines that are deficient in TCR and NER pathways. Our results revealed the existence of two apoptotic modes at low UV (2-4J/m2) doses in TCR deficient (UV61) and NER deficient (UV5) cells: one occurring in the first G1 and the other in the second G1-phase following the first division. At high UV doses (8-32J/m2), UV61 and UV5 cells underwent apoptosis without entry into S-phase after a permanent arrest in the initial G1. In contrast to repair deficient cells, parental TCR proficient AA8 cells did not show a significant G1 arrest and apoptosis at doses below 8J/m2. UV61 (proficient in repair of 6-4 photoproducts (PPs)) and UV5 (deficient in 6-4 PP repair) cells showed similar patterns of cell cycle progression and apoptosis. Taken together, these results suggest that the persistence of 6-4 PP and the replication inhibition may not be critical for apoptotic response in hamster cells. Instead, the extent of transcription blockage resulting from the TCR deficiency constitutes the major determining factor for G1 arrest and apoptosis.

The WRN and MUS81 proteins limit cell death and genome instability following oncogene activation

Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.