H2AX is required for cell cycle arrest via the p53/p21 pathway

Phosphorylation of H2AX (gamma H2AX) is an early sign of DNA damage induced by replication stalling. However, the role of H2AX in the repair of this type of DNA damage is still unclear. In this study, we used an inactivated adeno-associated virus (AAV) to induce a stalled replication fork signal and investigate the function of gamma H2AX. The cellular response to AAV provides a unique model to study gamma H2AX function, because the infection causes pannuclear H2AX phosphorylation without any signs of damage to the host genome. We found that pannuclear gamma H2AX formation is a result of ATR overactivation and diffusion but is independent of ATM. The inhibition of H2AX with RNA interference or the use of H2AX-deficient cells showed that gamma H2AX is dispensable for the formation and maintenance of DNA repair foci induced by stalled replication. However, in the absence of H2AX, the AAV-containing cells showed proteosome-dependent degradation of p21, followed by caspase-dependent mitotic catastrophe. In contrast, H2AX-proficient cells as well as H2AX-complemented H2AX(-/-) cells reacted by increasing p21 levels and arresting the cell cycle. The results establish a new role for H2AX in the p53/p21 pathway and indicate that H2AX is required for p21-induced cell cycle arrest after replication stalling.

Viral transport of DNA damage that mimics a stalled replication fork

Peter Martin Beard, Andrzej Stasiak

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

2005

Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling

Peter Martin Beard, Michail Fragkos

Adeno-associated virus (AAV) DNA, by mimicking a stalled replication fork, provokes a DNA damage response that can arrest cells in the G2/M phase of the cell-cycle. This response depends strictly on DNA damage signaling kinases ATR and Chk1. Here, we used AAV to study long-term effects of DNA damage signaling in cells with altered p53 status. In HCT116 cells, in response to damage signaling, p53 represses transcription of the genes encoding mitotic regulators Cdc25C, cyclin B1, and Plk1 to establish a firm G2 arrest. Isogenic cells lacking p53 maintain these three proteins at constant levels yet can still arrest initially in G2 because Chk1 signaling inhibits their enzymatic activities. Unexpectedly, the levels of Chk1 fall abruptly in a proteasome-dependent manner after two days of arrest in G2. In p53-deficient cells, this Chk1 instability is coupled to recovery of the phosphatase activity of Cdc25C and in the kinase activities of Plk1 and Cdk1/cyclin B1. Consequently, the p53-deficient cells enter lethal mitosis. Thus, the Chk1-mediated arrest is transient: it initially causes cells to accumulate in G2 until p53-dependent transcriptional repression of mitotic proteins takes over. p53-deficient cells cannot maintain the DNA damage signaling-induced G2 arrest after Chk1 has disappeared, and continue into catastrophic mitosis. Restoring Chk1 prevents the cells from entering such mitosis. These results reveal a mechanism based on Chk1 stability that regulates mitotic entry after DNA damage and elucidate the controversial phenomenon of p53-promoted cell survival in the face of damage signaling.

2007

On the replication of adeno-associated virus in the presence or absence of viral helper functions

Anika Ekrut

Adeno-associated virus (AAV) is a small non-enveloped virus and as a member of the parvovirus family, it contains a single stranded DNA genome. AAV's life cycle in vitro is strongly dependent not only on cellular factors but also on helper functions provided by other viruses, for example adenovirus, herpes virus and, to a certain extent, papillomavirus (HPV). Although AAVs are wide spread throughout the human population, no disease is yet associated with them. Accordingly, AAV became attractive as a vector for gene therapy studies, but even though the virus is well studied in vitro and now used in clinical trials as a vector, not much is known about AAV infection in vivo. AAV was initially isolated from humans together with its helper adenovirus. However, adenovirus-independent replication of AAV has been reported for a number of cellular situations, including differentiating keratinocytes with or without the presence of HPV, and in cells with an activated DNA damage response due to genotoxic agents. In the work reported here we have undertaken a systematic study of these alternative helper activities with a view to evaluating their contribution to natural infections by AAV. We used three different systems of cultured keratinocytes and showed that in each case in vitro differentiation was achieved. In two of these, the cells were from HPV-induced lesions. We found that AAV replicates efficiently in differentiating monolayer foreskin keratinocytes in the presence but not in the absence of adenovirus. Likewise naturally HPV-infected cervical keratinocytes only support the full life cycle of AAV if adenovirus is present. Even though HPV can provide certain helper functions, it is less efficient as a helper than adenovirus. We also exploited osteosarcoma cells, which are particularly well infected by AAV. Our results indicate that U2OS osteosarcoma cells are semi-permissive for AAV growth. Induction of a DNA damage response in these cells, or in HPV-containing keratinocytes, stimulates AAV genome amplification. Although AAV replication in the absence of helper virus can readily be demonstrated, this is either at a relatively low level or limited to part of the AAV growth cycle. We conclude that although these alternative helper systems can contribute to the natural history of AAV, the presence of an efficient helper virus seems likely to be needed for AAV to persist in the human population.

EPFL2009

On the replication of adeno-associated virus in the presence or absence of viral helper functions

Anika Ekrut

EPFL2009