Cell Cycle and Genomic Stability

Vanesa Gottifredi - Fundación Instituto Leloir

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Cell Cycle and Genomic Stability

It is well known that the most frequent protocols used currently to treat cancer involve the use of chemotherapeutic drugs (combined or not with surgery). The cytotoxicity of most of those treatments is based on the direct or indirect generation of DNA replication defects which should mainly affect transformed cell with high proliferative potential. In this context, the study of mechanisms that regulate damaged DNA replication (tolerance, checkpoint and others) is central to identify key factors in tumor cell survival and chemoresistance. We have identified the cyclin kinase inhibitor p21 as the first global negative regulator of the tolerance process called translesion DNA synthesis (TLS). P21 is a potent inhibitor of the recruitment to damaged DNA of all polymerases in the Y family. P21 also controls the progression of DNA replication forks at DNA lesions and its downregulation is required to maintain the genomic stability after UV irradiation. We propose that p21 is a central regulator of mutagenesis processes associated to DNA replication and it is therefore a protein with multiple functions during different phases of the cell cycle. We have also found that a protein in charge of the detection of replication defects, the kinase Chk1, also coordinates DNA replication and TLS. The information generated by us might be of use in the design/reformulation of combined therapies.

Background

DNA lesions are frequent events which are triggered by exogenous but also endogenous metabolic intermediates. The topological changes provoked by those lesions can irreversibly block DNA replication which in turn can lead to cell death. This is why the recruitment of tolerance factors to DNA lesions is important to promote cell survival and avoid the collapse of stalled replication forks. Unfortunately, this could represent a double edge sward and, within a pathological context could promote increased chemoresistance associated with increased mutagenesis. Therefore, the identification of novel regulators of tolerance mechanisms can be extremely useful for the design of alternative cancer therapies.

Goals

Our questions are the following:
  • Which are the central factors that coordinate DNA replication, DNA repair and the tolerance of DNA lesions?
  • How is the recruitment of tolerance factors spacio-temporally limited to damaged DNA?
  • Can the tolerance mechanisms been negatively regulated in tumor cells to achieve more efficient cell killing?

Methodology

We can group our experimental approaches in 3 categories:
  • The analysis of protein-protein interactions: the techniques routinely used in our laboratory include the determination of proteins half-life, identification and quantification of degradative and non-degradative ubiquitination, the evaluation of protein-protein interaction in the chromatin fraction, the transient modulation of proteins levels by overexpression or siRNA driven negative regulation.
  • The study of subnuclear localization and the mobility of proteins in living cells: we monitor the modification in the nuclear organization of proteins adapting the quantification criteria that better describes the type and strength of the modification. We apply global and local UV irradiation techniques using polycarbonate filter and alpha particle irradiation. We also study the mobility of fluorescent tagged proteins in living cells. The latter techniques include a spacial analysis and the utilization of two photon microscopy.
  • the exploration of the biological impact on the overall cellular response to DNA damage: we routinely perform cell cycle analysis using FACS technology. To assess the global effect on TLS we apply a technique called DNA stretching that determines replication rates in single DNA molecules. We also determine mutation frequency and mutation spectrum associated to DNA damage tolerance.

Speroni J, Federico MB, Mansilla S, Soria G and Gottifredi V. (2012). A kinase independent role of Chk1 in the replication of damaged DNA. Proc Natl Acad Sci U S A. May 8, 109(19) 7344-9. PubMed

Soria, G., Gottifredi. V. PCNA-coupled p21 degradation after DNA damage: and exception that confirms the rule?DNA repair. 9: 358-364 (2010).     PubMed

Soria, G., Belluscio, L., van Cappellen, W.A., Kanaar, R., Essers, J., Gottifredi, V. DNA damage induced Pol eta recruitment takes place independently of the cell cycle phase. Cell Cycle. 8: 3340-3348 (2009).     PubMed

Prives, C., Gottifredi, V. The p21 and PCNA partnership: a new twist for an old plot. Cell Cycle. 7: 3840-3846 (2008).     PubMed

Soria, G., Speroni, J., Podhajcer, O.L., Prives, C., Gottifredi, V. p21 differentially regulates DNA replication and DNA-repair-associated processes after UV irradiation. J Cell Sci. 121: 3271-3282 (2008).     PubMed

Soria, G., Podhajcer, O., Prives, C., Gottifredi, V. P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation. Oncogene. 25: 2829-2838 (2006).     PubMed

Gottifredi, V., Prives, C. The S phase checkpoint: when the crowd meets at the fork. Semin Cell Dev Biol. 16: 355-368 (2005).     PubMed

Gottifredi, V., McKinney, K., Poyurovsky, M. V., and Prives, C. (2004). Decreased p21 levels are required for efficient restart of DNA synthesis after S phase block. J Biol Chem 279, 5802-5810. PubMed

Gottifredi, V., and Prives, C. (2001). Molecular biology. Getting p53 out of the nucleus. Science 292, 1851-1852. IF: 23,32 PubMed

Gottifredi, V., Shieh, S., Taya, Y., and Prives, C. (2001). p53 accumulates but is functionally impaired when DNA synthesis is blocked. Proc Natl Acad Sci U S A 98, 1036-1041. PubMed

Gottifredi, V., Karni-Schmidt, O., Shieh, S. S., and Prives, C. (2001). p53 down-regulates CHK1 through p21 and the retinoblastoma protein. Mol Cell Biol 21, 1066-1076. PubMed





Vanesa Gottifredi
Head of Laboratory - vgottifredi@leloir.org.ar



Sabrina Mansilla
Research Assistant - CONICET



Marina Alejandra González Besteiro
Research Assitant - CONICET



Julieta Martino
Posdoctoral Fellow - ANPCyT



Natalia Soledad Paviolo
Posdoctoral Fellow - ANPCyT



Nicolás Calzetta
PhD Fellow - CONICET



María Belén de la Vega Paéz
PhD Fellow- CONICET



Sofía Venerus Arbilla
PhD Fellow- AGENCIA



Sofia Loureiro
Predoctoral Fellow



Victoria Pauwels
Predoctoral Fellow