Atemin, A. S. | bioRxiv Science

bioRxiv2024

In and out of replication stress: PCNA/RPA1-based dynamics of fork stalling and restart in the same cell

Replication forks encounter various impediments which induce fork stalling and threaten genome stability, yet the precise dynamics of fork stalling and restart at the single-cell level remain elusive. Herein, we devise a live-cell microscopy-based approach to follow hydroxyurea-induced fork stalling and subsequent restart at 30-s resolution. We measure two distinct processes during fork stalling. One is rapid PCNA removal, which reflects the drop in DNA synthesis. The other is gradual RPA1 accumulation on up to 2400 nt of ssDNA per fork despite an active intra-S checkpoint. Restoring the nucleotide pool enables prompt restart without post-replicative ssDNA and a smooth cell cycle progression. ATR, but not ATM inhibition, accelerates hydroxyurea-induced RPA1 accumulation nine-fold, leading to RPA1 exhaustion within 20 min. Fork restart under ATR inhibition led to the persistence of [~]600 nt ssDNA per fork after S-phase, which reached 2500 nt under ATR/ATM co-inhibition, with both scenarios leading to mitotic catastrophe. MRE11 inhibition had no effect on PCNA/RPA1 dynamics regardless of ATR activity. E3 ligase RAD18 was recruited at stalled replication forks in parallel to PCNA removal. Our results shed light on fork dynamics during nucleotide depletion and provide a valuable tool for interrogating the effects of replication stress-inducing anti-cancer agents.

molecular biology↗

bioRxiv2024

Kinetic landscape of single virus-like particles highlights the efficacy of SARS-Cov-2 internalization

The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression, pH change, and nucleocapsid release. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into Rab5-negative endosomes via Dynamin-dependent scission. Less than two minutes later, the pH of VLPs drops below 5 followed by an increase in VLP speed, yet these two events are not interrelated. Nucleocapsid release from the VLPs occurs with similar kinetics to the pH drop, suggesting that VLP fusion occurs during endosome acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site altered the rate of VLP internalization events, indicating that they do not affect these processes. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells. Time-lapse videos of the studied internalization events can be accessed in the dedicated COVIDynamics database.

cell biology↗