Sathyanarayana, P. | Search bioRxiv

bioRxiv2025

Mechanisms of HSV-1 helicase-primase inhibition and replication fork complex assembly

Herpesviruses are widespread double-stranded DNA viruses that establish lifelong latency and cause various diseases. Although DNA polymerase-targeting antivirals are effective, increasing drug resistance underscores the need for alternatives. Helicase-primase inhibitors (HPIs) are promising antivirals, but their mechanisms of action are poorly defined. Furthermore, how the helicase-primase (H/P) complex and DNA polymerase coordinate genome replication is not well understood for herpesviruses. Here, we report cryo-EM structures of the herpes simplex virus (HSV) H/P complex bound to HPIs, showing that these lock the helicase-primase complex in an inactive conformation. Single-molecule assays reveal that HPIs cause helicase-primase complexes to pause in unwinding activity on DNA. The structure of an HPI-bound replication fork complex, comprising the H/P complex (UL5, UL52, and UL8) and polymerase holoenzyme (UL30 and UL42), reveals a previously uncharacterized interface bridging these complexes. These findings provide a structural framework for understanding herpesvirus replisome assembly and advancing inhibitor development.

microbiology↗

bioRxiv2025

The NHEJ machinery is translocated off DNA ends toenable resection

Error-prone non-homologous end joining (NHEJ) and homologous recombination (HR) compete to repair DNA double strand breaks [1, 2, 3]. Given that NHEJ factors such as Ku and DNA-PKcs are rapidly recruited to DSBs and sterically occlude ends, they must be removed to enable 5' DNA end resection, the commitment step of HR. While resection is well characterized, how the NHEJ machinery is displaced from DNA ends has remained poorly understood. Here, we performed single-molecule imaging in Xenopus laevis egg extracts to monitor the removal of NHEJ factors from DNA ends while simultaneously tracking the progress of DNA end resection. We find that MRN and CtIP are essential to evict the core NHEJ factor Ku from DNA ends. Surprisingly, while Ku removal and resection of the 5' end are normally coordinated, these processes arise from distinct activities of the MRN complex and are differentially regulated by the action of cyclin dependent kinases and ATM. By directly imaging the position of Ku on DNA using smFRET, we found that Ku is translocated independently of DNA end resection in an ATP-dependent manner that requires both MRN and CtIP. In summary, our results provide a new mechanistic framework to understand how eviction of the NHEJ machinery is coordinated with DNA resection to initiate HR.

biophysics↗