Involvement of the translesion synthesis polymerase η in the replication of G4s

In the last decade, new roles have been emerging for TLS polymerases. Among these specialized enzymes, polη represent the most eclectic member, playing a pivotal role in the bypass of UV-induced damage, contributing to the replication of intrinsically difficult to replicate regions and exerting a reverse transcriptase activity that suggests its involvement in RNA:DNA hybrids metabolism. Moreover, polη has been suspected of participating in G4s metabolism for a long time. It is able to replicate across G4s in vitro more efficiently and faithfully than polε and its depletion sensitizes cells to the G4s stabilizing compound telomestatin. G4s represent a huge obstacle for the replicative fork progression since they cannot fit in the active sites of canonical polymerases, thus requiring their resolution before they can cause their stalling. G4s unwinding is mediated by many helicases that are thought to work in multiple redundant pathways, one of which includes the TLS polymerase Rev1. Nevertheless, the replication across these structures still remains unclear. We decided to investigate whether the catalytic activity of polη observed on G4s substrates in vitro was recapitulated in vivo, exploiting a cellular isogenic model composed of XPV-patient derived fibroblasts, which lack polη, and a series of polη complemented clones. We used the G4s stabilizing compound PDS to better highlight the differences in G4s metabolism based on the expression on polη. We found that cells lacking polη are more sensitive to PDS and accumulate a greater amount of stabilized G4s. For the first time, we show that poη interacts physically to G4 containig DNA and its catalytic activity is essential to avoid G4s spreading after PDS. The PIP and UBZ domains are dispensable for polη recruitment at G4s, but they seem to mediate interactions that restrain polη catalytic activity on G4s and protect it from degradation. We observed a role of polη in regulating DNA replication and cell cycle progression upon G4s stabilization, alongside the induction of different DDR pathways based on the presence of the polymerase. Finally, we identified other TLS polymerases bound to G4s in vivo, possibly suggesting a more general involvement of TLS in G4s metabolism.