Investigating the Role of Fork Reversal Enzymes in G-quadruplex Resolution within the Terminal Repeats of Kaposi Sarcoma-associated Herpesvirus

Fork reversal is a protective mechanism that preserves genome integrity by stabilizing stalled and uncoupled replication forks. Fork reversal enzymes are hypothesized to promote bypass of DNA secondary structures, but their function in G-quadruplex (G4s) resolution remains poorly characterized. In this study, we investigated the role of fork reversal in resolving G4s within the terminal repeat (TR) region of the oncogenic Kaposi Sarcoma-associated Herpesvirus (KSHV). The genome of KSHV contains an 801 bp G-rich sequence tandemly repeated 30-50 times, which is predicted to form G4s and is essential for viral maintenance. Using Xenopus laevis egg extracts, we performed plasmid pulldown and immunodepletion assays targeting key fork reversal factors, namely SMARCAL1, ZRANB3 and HLTF, and the nuclease DNA2. Plasmid pulldown analysis showed an enrichment of fork reversal proteins on TR-containing plasmids during DNA replication, particularly in the presence of G4 stabilizing small molecule PhenDC3. However, immunodepletion assays of these enzymes did not reveal a significant inhibition of DNA replication kinetics. Altogether, our findings indicate that fork reversal proteins are recruited to G4-rich sequences, but their functional role might be redundant with that of alternative G4 resolution mechanisms. Additionally, they underline the complexity of KSHV genome maintenance and suggest that a multi-pathway inhibitory strategy may be required to successfully disrupt viral replication.