Given the mutagenic potential of DNA lesions, the mechanisms in charge of the removal of damaged bases or the elimination of genetically unstable cells constitute our main lines of defense against cancer. However, the excessive removal of cells with acceptable levels of DNA damage is also detrimental for multicellular organisms. In that context it is important to consider that the cells are particularly sensitive when DNA replication takes place since cell death can be triggered by the irreversibly stall of DNA polymerases (Pols) at DNA lesions. In such scenario auxiliary events know as DNA-damage tolerance mechanism promote DNA replication and insofar, avoid replication fork collapse and protect cell viability. A key DNA damage tolerance mechanism is called Translesion DNA synthesis (TLS). When replicative DNA Pols stop at DNA lesions, a family of specialized DNA Pols, characterized by their relaxed fidelity, can synthesize DNA opposite such lesions. In that way, the processivity of replication forks is maintained and cell death is avoided. However, TLS Pols are mutagenic when compared to replicative counterparts. Thus, it is very likely that negative regulators of TLS polymerases have the important function of inhibiting their recruitment to undamaged DNA templates in order to avoid unnecessary mutagenesis. The induction of TLS after genotoxic challenge can be studied by monitoring convenient markers such as PCNA ubiquitination, the increase in focal organization of specialized Pols and the increase in the interaction of PCNA and specialized Pols. Our data, which was obtained in collaboration with Carol Prives in Columbia University and supported by the FIRCA RO3TW007440, indicates that the cyclin kinase inhibitor p21 is a potent negative regulator of those TLS features. Interestingly, we have observed that many genotoxic treatments that require TLS activation strongly increase p21 proteolysis, being this effect dominant on p53 transactivation. Moreover, we have also observed that basal p21 levels detected in unstressed conditions also exert a negative effect on the TLS markers discussed above. Taking together our data we hypothesized that p21 might work as one important switch that represses the activity of specialized Pols on undamaged DNA but releases them to a full activation mode when DNA lesions are encountered by the replication fork. To test our hypothesis, we conceived two main aims: the first one is to determine the impact of UV-induced p21 degradation on the proper replication of damaged DNA (which depends on full TLS activation);
the second aim will explore the effect of basal p21 on the control of specialized Pols activity during unstressed replication. To do so we will combing Flow Cytometry analysis with more specific techniques such as DNA combing technology, different mutagenesis assays, cytogenetic and FISH technology. Our proposal will serve to identify the exact contribution of p21 to the control of the delicate balance between DNA damage tolerance and mutagenesis.

Public Health Relevance

DNA damage must be removed but, during the replicative phase of the cell cycle, it must also be used as a template for DNA duplication. Specialized DNA polymerases characterized by low fidelity must perform this task and therefore, they must be tightly control to avoid unnecessary mutagenesis. We have obtained preliminary evidence indicates that the p21 cyclin kinase inhibitor is a potent negative regulator of specialized DNA polymerases and it is our goal to explore its effect on the replication of damaged DNA.

National Institute of Health (NIH)
Fogarty International Center (FIC)
Small Research Grants (R03)
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International and Cooperative Projects - 1 Study Section (ICP1)
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Light, Enid
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Fundacion Instituto Leloir
Buenos Aires
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Mansilla, Sabrina F; Soria, Gaston; Vallerga, Maria Belen et al. (2013) UV-triggered p21 degradation facilitates damaged-DNA replication and preserves genomic stability. Nucleic Acids Res 41:6942-51
Costantino, Valeria V; Mansilla, Sabrina F; Speroni, Juliana et al. (2013) The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers. PLoS One 8:e53168
Speroni, Juliana; Federico, Maria Belen; Mansilla, Sabrina F et al. (2012) Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA. Proc Natl Acad Sci U S A 109:7344-9