Phd in HUMAN BIOLOGY AND MEDICAL GENETICS

Thesis title: The multiple roles of WRN protein in replication stress tolerance

DNA replication is a challenging process for eukaryotic cells, replication forks (RFs) are frequently threatened and arrested by intrinsic RF obstacles such as transcribing RNA polymerases, unusual DNA structures, tightly bound protein-DNA complexes or oncogene activation in addition to DNA lesions induced by endogenous or exogenous agents. To maintain genome stability eukaryotic cells developed multiple mechanisms to replicate DNA correctly, avoiding inheritable DNA lesions such as point mutations or chromosomal rearrangements. These mechanisms are orchestrated by the S-phase checkpoint under the handling of ATR. Indeed, several of the proteins involved in stalled-fork remodelling are regulated by ATR, including the RECQ helicase WRN. The absence of this protein, due to biallelic mutations of the WRN gene, causes a rare genetic disorder known as Werner syndrome (WS). WS patients typically exhibit a range of symptoms, including premature aging and an increased risk of certain types of cancer that reflect the importance of the WRN protein in maintaining genomic stability and repairing DNA damage. Our aim was to study the poorly understood molecular mechanism through which ATR regulates the activity of WRN during the processing of stalled forks in order to clarify how perturbed DNA replication can be overcome. Performing a combination of biological experiments using genetically defined human cell lines, I found that loss of WRN phosphorylation by ATR results in a deregulated nascent DNA degradation at stalled forks in response to replication perturbation induced by a short treatment with the drug hydroxyurea (HU), which inhibits the activity of ribonucleotide reductase thereby depleting the cellular nucleotide pool. HU exposure is sufficient to trigger fork arrest and regression but not double-strand breaks (DSBs) in wild-type cells. Interestingly, the WRN mutations abrogating phosphorylation enhanced DSBs formation dependent on two structure-specific nucleases, MUS81 and MRE11, probably related to the decreased fork protection activity of the ssDNA binding protein RAD51, mimicking the pathological condition observed in BRCA-deficient cells. The outcome is an increased genomic instability when WRN is not regulated by the ATR kinase. The expression of WRN containing mutations mimicking a constitutive phosphorylation by ATR channels reversed fork through a recombination dependent fork restart bypassing DSBs completely. Strand invasion behind the stalled fork causes the accumulation of the DNA translocase ZRANB3, which is fundamental to maintain genome stability, probably mediating the resolution of a D-loop structure downstream of RAD51. Furthermore, experiments involving chemical inhibition of WRN helicase demonstrated that this function is deregulated when ATR phosphorylation is persistent in the WRN-phosphomimetic expressing cells. Taken together my results show that the regulation of WRN mediated by ATR activates the canonical pathways for the handling of stalled forks but leads to engagement of a previously unidentified RAD51-ZRANB3 pathway if the phosphorylation is not finely tuned. In addition to defining the framework of the ATR-dependent regulation of WRN, my work aimed to define the elusive replicative function of the WRN exonuclease enzymatic activity. My studies provides evidence that loss of WRN exonuclease leads to accumulation of single-stranded DNA (ssDNA) gaps and S phase poly(ADP-ribose) foci, a marker of unligated Okazaki fragments (OFs). In addition, our data show that WRN exonuclease-defective cells are resistant to inhibition of FEN1, further supporting the hypothesis that an alternative mechanism of Okazaki fragments maturation (OFM) is engaged. However, more studies are required to define the underlying molecular mechanism. Overall, this study implicates WRN and its ATR-mediated regulation as a crucial factor in the restart of perturbed replication fork. Under conditions of replication stress WRN regulation by ATR is required for a timely selection of the pathway deputed to restart replication, allowing cells to proliferate and granting genome stability. Our studies indicate that WRN, through its helicase activity and the ATR- dependent regulation, stabilises reversed forks preventing degeneration into DSBs, and through its exonuclease activity contributes to proper metabolism of the lagging strand. These two functions, either collaborating or acting differently during perturbed vs. unperturbed replication, could be critical for genome stability but might also be relevant for cancer research. WRN is a potential druggable target and defining whether the helicase activity of WRN is regulated by ATR would be interesting to evaluate WRN-helicase inhibitors for potential combinatory treatments of tumors acquiring resistance to ATR inhibitors. In addition, targeting WRN exonuclease can be explored for combinatorial therapy in tumors with defective metabolisms of the lagging strand.