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The encounter of a replication fork with a blocking DNA lesion is a common event that cells need to address properly to preserve genome integrity and to avoid consequences such as Cancer. Many proteins are involved in blocked fork stabilization as for example, BRCA2, an important tumour susceptibility gene that has recently been described as having a key role in preventing fork collapse. Stalled replication forks cause the formation of daughter-strand gap that are subsequently filled-in by pathways referred to as post-replication repair or tolerance pathways. However, despite being a widely used notion, little is known in vivo about the fate of a defined blocked replication fork by lack of appropriate methodology. We report a novel methodology to analyze the consequences of a single replication-blocking lesion site-specifically located in the E. coli genome. Both Translesion Synthesis (TLS) and Damage Avoidance (DA) events are monitored. Inactivation of recA strongly decreases the occurrence of DA events, independently of the lesion being located in the leading or lagging strand. While, under physiological conditions, TLS events are rare compared to DA events, modest over-expression of TLS polymerases strongly enhances TLS, suggesting that TLS polymerase concentrations represent the rate-limiting step. We conclude that cells implement tolerance pathways in a defined order, TLS first followed by DA.