SPARTAN promotes genetic diversification of the immunoglobulin-variable gene locus in avian DT40 cells

Prolonged replication arrest on damaged templates is a cause of fork collapse, potentially resulting in genome instability. Arrested replication is rescued by translesion DNA synthesis (TLS) and homologous recombination (HR)-mediated template switching. SPARTAN, a ubiquitin-PCNA-interacting regulator, regulates TLS via mechanisms incompletely understood. Here we show that SPARTAN promotes diversification of the chicken DT40 immunoglobulin-variable λ gene by facilitating TLS-mediated hypermutation and template switch-mediated gene-conversion, both induced by replication blocks at abasic sites. SPARTAN-/- and SPARTAN-/-/Polη-/-/Polζ-/- cells showed defective and similar decrease in hypermutation rates, as well as alterations in the mutation spectra, with decreased dG-to-dC transversions and increased dG-to-dA transitions. Strikingly, SPARTAN-/- cells also showed reduced template switch-mediated gene-conversion at the immunoglobulin locus, while being proficient in HR-mediated double strand break repair, and sister chromatid recombination. Notably, SPARTAN's ubiquitin-binding zinc-finger 4 domain, but not the PCNA interacting peptide domain or its DNA-binding domain, was specifically required for the promotion of immunoglobulin gene-conversion, while all these three domains were shown to contribute similarly to TLS. In all, our results suggest that SPARTAN mediates TLS in concert with the Polη-Polζ pathway and that it facilitates HR-mediated template switching at a subset of stalled replication forks, potentially by interacting with unknown ubiquitinated proteins.