The genome of every living cell is under constant attack from agents that compromise the chemical integrity of DNA. Most of these lesions are removed by efficient repair mechanisms, but some escape this surveillance and block the progression of the replicative DNA polymerase. When a replication fork encounters such an unrepaired lesion, the cell has two distinct tolerance pathways at its disposal. Translesion synthesis, or TLS, relies on specialized polymerases capable of inserting a nucleotide directly opposite the lesion, at the cost of a high mutational risk. Conversely, damage avoidance relies on faithful homologous recombination, which fills in the single-stranded DNA region generated downstream of the lesion. The balance between these two pathways is critical: it sets the level of mutagenesis during lesion bypass, a source of both adaptive genetic variability and risk of genome instability.
Using a system that allows simultaneous monitoring of TLS and recombination-mediated repair at a single lesion inserted in a site-specific manner into the *Escherichia coli* chromosome, the authors show that a hitherto overlooked factor strongly modulates this balance: the mere proximity of two blocking lesions located on opposite strands. When two lesions are close together, the single-stranded DNA regions generated downstream of each overlap, which locally inhibits homologous recombination and triggers an increased reliance on the more mutagenic TLS. By varying the spacing between lesions and exploiting mutant strains, the team estimates that the initial gap generated downstream of a lesion extends over 1.8 to 3.5 kilobases. The RecJ exonuclease enlarges these gaps: beneficial at low lesion density because it promotes recombination, its action becomes deleterious at high density by causing the single-stranded regions to overlap.
This structural inhibition of recombination proves to be independent of the other known regulatory mechanisms. It is distinct from the genetic inactivation of *recF*, which reduces RecA loading, and their effects are additive: the combination leads to a more than sevenfold increase in TLS. Likewise, the effect is independent of SOS response activation, but additive with it. Induction of the SOS system increases TLS usage by approximately fivefold, and lesion proximity adds a further twofold factor: when lesions are close together and SOS is induced, error-prone TLS accounts for nearly 90% of survival. This effect persists in a context proficient for nucleotide excision repair. The authors propose that this structural mechanism, acting independently of genetic factors, constitutes a new paradigm of mutagenesis induced by genotoxic stress, potentially transferable to other organisms given the conservation of homologous recombination across species.