Study of the functions of mammalian primpol protein in vivo

Resumen

During eukaryotic DNA replication, the replisome machinery slows down when the template DNA is damaged by endogenous or exogenous elements. To minimize the risk of fork collapse, “DNA damage tolerance” (DDT) mechanisms facilitate progression of replication through damaged templates. A classical DDT response involves the use of DNA polymerases specialized in “translesion synthesis”. An alternative DDT mechanism consists in the reinitiation of DNA synthesis downstream of the damaged DNA, leaving a short unreplicated gap to be repaired post-replicatively. This mechanism requires the repositioning of the replisome and a new priming event. In 2013, mammalian PrimPol protein was identified as the enzyme that mediates fork restart after different types of DNA lesions, including thymine photodimers caused by UV light. PrimPol is a member of the archaeal-eukaryotic primase (AEP) superfamily and is included in a clade of AEP proteins that contain both primase and polymerase activities. Recent studies have confirmed a similar role for PrimPol at natural obstacles or difficult-to-replicate sites. Despite these antecedents, the contribution of PrimPol to maintaining genomic stability and its possible protective function against UV-induced DNA damage remained to be investigated. In this thesis we have studied the functions of mouse PrimPol in vivo. In a first approach, we participated in a structure-function study designed to understand the functional importance of several conserved amino acids in the PrimPol family, including two variants presumably associated to pathological conditions: Y89D and Y100H. This study led to the identification of two amino acids located within a conserved WFYY motif that are indispensable for PrimPol functionality. In the second part of the thesis, we have studied the physiological consequences of PrimPol ablation in mammals using a KO mouse model. PrimPol-/- mice are viable but hypersensitive to UV-induced DNA damage. Without PrimPol, skin healing after UV irradiation is compromised due to inefficient cell proliferation in the epidermis, which is necessary to replace cells with damaged DNA. The absence of PrimPol also caused a higher incidence of UV-induced papillomas, gender-specific alopecia, obesity and the development of aggressive tumors. Our results underscore the importance of mammalian PrimPol for the maintenance of genomic stability and strongly suggest a tumor suppression function in vivo.