Giorgio Baldi

The DNA replication machinery can encounter many obstacles while duplicating the genome, which put the replicative apparatus under stress. Cells have developed numerous mechanisms to overcome replication stress (RS). After the loss of key factors, that help to deal with RS, deleterious events can occur. The main pathway implicated in DNA damage response is DNA damage checkpoint. When DNA replication is blocked, checkpoint activation ensures structural stability of the replisome avoiding fork collapse and promoting DNA replication resumption. One of the hallmarks of RS is single-stranded DNA accumulation at the replication forks. Here we used AFM and EM to provide a detailed characterization of DNA lesions arising in RS conditions imposed by DNA polymerase inhibitors in Xenopus laevis egg extract. We identified different intermediates induced by RS, including ssDNA gaps and reversed forks (RVFs). Importantly, we directly correlated the presence ssDNA gaps with the onset RVFs. We identified one possible source of ssDNA gap accumulation at forks by showing that homologous recombination protein Rad51 is required for optimal function of Pola at stressed replication forks. Rad51-Pola interaction is likely to be important for stalled fork resumption. We also provided evidence that replication fork with ssDNA gaps are converted into RVFs by Smarcal1. We also showed RVFs can trigger Mre11-dependent DNA degradation upon RS in the absence of Rad51. We provided mechanistic insights into checkpoint regulation of RVFs levels through ATR, Smarcal1 and Rad51 regulation. Overall this provides structural and molecular insights into the metabolisms of replication forks under stressful conditions.