IDENTIFICATION AND CHARACTERIZATION OF STRESSED REPLICATION FORK INTERMEDIATES

The DNA replication machinery encounters a number of obstacles while duplicating the genome, which put the replicative apparatus under stress. Cells have developed a number of mechanisms to overcome replication stress sources. However, in the presence of chronic stress, or after loss of key factors that help to deal with this stress, a range of deleterious events can occur. One of the main pathways implicated in DNA damage sensing and response is DNA damage checkpoint. When DNA replication fork progression is blocked, checkpoint activation ensures structural stability of the replisome components avoiding fork collapse and promoting, when possible, fast resumption of DNA replication. One of the hallmarks of replication stress is the accumulation single-stranded DNA at the replication forks. Here we used Atomic Force microscopy (AFM) and Electron Microscopy (EM)-based approaches to provide a detailed characterization of DNA lesions arising in the presence of replication stress imposed by interference with DNA polymerase activity in Xenopus laevis egg extract. We identified a number of intermediates induced by DNA polymerase inhibition, including replication forks containing ssDNA gaps and reversed forks (RVFs). Importantly, we directly correlated the presence and the length of ssDNA gaps at the replication fork junctions with the onset RVFs. Significantly, we identified one possible source of ssDNA gap accumulation at forks by showing that homologous recombination protein Rad51 is required for optimal function of Polymerase-alpha at stressed replication forks. To fulfill this function, Rad5/Pol-alpha interaction is likely to be important for stalled fork resumption. We also provided evidence that replication fork intermediates with persistent ssDNA gaps are converted into RVFs by Smarcal1 translocase, showing that this enzyme is among the most important fork remodelers that can trigger fork reversal upon fork stalling. We also showed that RVFs can trigger extensive Mre11 dependent DNA degradation upon replication stress in the absence of functional Rad51. Finally, 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.