Mechanisms of translesion synthesis in human cells

Abstract

Ultraviolet (UV) light is a highly carcinogenic agent to which the human is exposed, that damages the cellular components and deforms the DNA strand and can result in cell death or gene instability. The lesions formed by UV light are usually removed by the Nucleotide Excision Repair mechanism (NER). When these lesions remain until the replication, there is a process known as translesion synthesis (TLS), a mechanism of tolerance to damage performed by specific polymerases. This mechanism allows the cell to continue replication and is partially under the control of the PCNA protein, which normally acts as a platform for DNA polymerases. There are two models proposed to explain how the TLS process occurs: directly after the replication fork blockage or by the gap-filling model. These tolerance mechanisms may occur through the action of one or more TLS polymerases (Pols). Among them, Pol eta (·) is the most studied and its absence leads to a milder phenotype of xeroderma pigmentosum (XP) syndrome, the variant form (XP-V). However, it is not yet known which TLS is involved in the tolerance of photoproducts in the absence of Pol·, leaving open an issue that touches directly on the explanation of mutagenesis in XP-V patients. It is known that Pol iota (¹), another Pol of TLS, is found together with Pol· in replication foci. In addition, we have recently found that Pol· induction is an important mechanism responsible for increasing cell survival of primary fibroblasts (NER deficient) after pre-treatment with low doses of UVC irradiation followed by irradiation at higher doses. However, this does not occur for primary fibroblasts deficient in Transcription Coupled Repair (TCR), perhaps due to structures formed after the transcription blockade by lesions, the R-loops. In this context, the objectives of the present project are to investigate the role of Pol¹ in the bypass of UV-induced photoproducts in repair-deficient human cells and to evaluate the effect of Pol· induction on TCR-deficient primary cells to understand how the induced replication has the TLS affected in this cells. For this, we will use cell lines silenced for Pol¹ and/or Pol· previously established in our laboratory, as well as primary fibroblasts from human patients with mutations in the XP-A and CSB genes. In addition, we also intend to characterize a cell line (from a patient with clinical phenotypes related to neurodegeneration) newly established in our laboratory with a new mutation in the gene encoding PCNA, investigating the consequence of this mutation on repair and tolerance processes of lesions in the genome. (AU)