Study of PrimPol function at DNA interstrand crosslinks

During genomic replication, damaged DNA templates and difficult-to-replicate DNA structures slow down the progression of replication forks, a situation referred to as replication stress (RS). The worst possible outcome of RS is the collapse of forks, which generates double strand breaks. This is prevented by DNA damage tolerance (DDT) mechanisms that facilitate replication through damaged DNA or other replication blocks. DDT allows continued DNA synthesis, leaving the damaged template to be fixed by post-replicative DNA repair mechanisms. DNA interstrand crosslinks (ICLs) consist of covalent bonds between the two complementary DNA strands and were initially assumed to be absolute blocks for the DNA replication machinery. However, recent work has discovered that ICLs may be bypassed by replication forks, a process that has been termed “ICL traverse” and remains poorly understood at the mechanistic level. ICL repair requires DNA replication and a combination of nucleotide excision repair (NER), homologous recombination (HR) and translesion synthesis (TLS). Failure to repair ICLs results in Fanconi Anemia (FA), a rare but very severe disease. PrimPol, the second primase in human cells after the canonical Pol a-primase complex, was characterized in 2013 as a major player in DDT. Upon UV-induced DNA damage, PrimPol is recruited to stalled forks and synthesizes new DNA primers downstream of DNA photoadducts, facilitating re-initiation of DNA synthesis and leaving behind short unreplicated gaps to be repaired at a later time. In this Thesis we have studied the participation of human PrimPol in the cellular response to ICLs. We describe that PrimPol is relocalized to chromatin upon ICL generation and uses its primase activity to facilitate ICL traverse, which in turn creates a suitable template for subsequent ICL repair. The ablation of PRIMPOL by Crispr/Cas genetic editing resulted in inefficient ICL repair, increased chromosomal instability, and cellular dependence on an alternative mechanism to initiate ICL repair that relies on the convergence of two forks at the lesion. The loss of PrimPol also resulted in “synthetic sickness” phenotypes with the downregulation of the FA pathway and proteins involved in fork stabilization and DNA repair. PRIMPOL KO cells and mice were hypersensitive to ICL-inducing agents, opening the exciting possibility of targeting PrimPol activity to enhance the efficacy of cancer therapy based on DNA crosslinking agents