RecQL helicases represent a unique family of conserved proteins. The five human family members share the characteristic helicase core domains that confer the 3’-5’ DNA helicase activity. Non-conserved domains provide substrate specificity, different protein interactions and ultimately result in the varying functions of each homolog. In this thesis we focus on the synthetic lethal interaction of WRN in the context of microsatellite instability and the role of RECQL4 helicase in DNA origin firing.

Genetic loss or inhibition of WRN leads to severe reduction in viability of cancer cells exhibiting Microsatellite Instability (MSI). Studies point to the helicase activity of WRN as the contributing factor of MSI cell survival. Mechanistically, WRN is required for the resolution of putative secondary DNA structures associated with the expansion of DNA microsatellite elements. Such structures pose a significant threat to DNA replication, acting as ‘roadblocks’ to replisomes and leading to the generation of DNA damage and chromosomal instability. Yet, the existence of MSI cell lines insensitive to WRN inhibition implies that additional functions of WRN may contribute to the synthetic lethality. In this study we demonstrate that WRN is suppressing the generation of DNA damage both in and out of the S-phase in MSI cells. We show that WRN-sensitive and -insensitive MSI cells exhibit different patterns of DNA damage upon inhibition of WRN. In addition, we describe the unique nature of the cell cycle checkpoint activated in MSI cells treated with WRN inhibitors and identify similarities with the putative TOPOII-related DNA decatenation checkpoint. Furthermore, we showcase the different response of WRN-sensitive and -insensitive MSI cells to concomitant inhibition of WRN and TOPOII, suggesting the relevance of the interplay of WRN and TOPOII in MSI cells. Altogether, we reveal that different cellular functions of WRN are responsible for the synthetic lethality of WRN in MSI cells, paving the way for the identification of novel therapeutic targets.

The unique N-terminal domain of RECQL4 shares sequence similarity with the yeast Sld2 protein, an essential factor for DNA origin firing. A number of studies describe the implication of RECQL4 homologs from different organisms in DNA replication initiation. Yet, the exact functions of human RECQL4 in origin firing remain elusive. In our study we demonstrate that human RECQL4 WT and CRISPR-Cas9-generated knock-out (KO) cells exhibit similar S-phase entry kinetics and unaltered patterns in origin firing. Single-molecule experiments show that RECQL4 affects replication fork progression in a dominant negative manner, with KO clones displaying no defects in fork speed, while helicase-dead mutants exhibit slower fork progression rates. Interestingly, EdU-seq experiments revealed the genome-wide increase in mobility of the replication front in KO cells upon recovery from hydroxyurea, suggesting that RECQL4 might indirectly affect the progression of DNA replication under conditions of replication stress. Lastly, we show that RECQL4 does not participate in MiDAS, a BIR repair pathway responsible for rescuing under-replicated DNA regions in mitosis. Collectively, we provide evidence that RECQL4 has a dispensable role in DNA replication initiation under physiological conditions, while it regulates fork progression rates, showcasing its functional divergence from yeast Sld2.