During DNA synthesis, DNA replication and transcription machinery can collide, and the replication fork may temporarily dislodge RNA polymerase II (RNAPII) to resolve the transcription-replication conflict (TRC), a major source of endogenous DNA double-strand breaks (DSBs) and common fragile site (CFS) instability. However, the mechanism of TRC resolution remains unclear. Here, we show that conjugation of SUMO2, but not SUMO1 or SUMO3, to the essential replication factor PCNA is induced on transcribed chromatin by the RNAPII-bound helicase RECQ5. Proteomic analysis reveals that SUMO2-PCNA enriches histone chaperones CAF1 and FACT in the replication complex via interactions with their SUMO-interacting motifs. SUMO2-PCNA enhances CAF1-dependent histone deposition, which correlates with increased histone H3.1 at CFSs and repressive histone marks in the chromatin to reduce chromatin accessibility. Hence, SUMO2-PCNA dislodges RNAPII at CFSs, and overexpressing either SUMO2-PCNA or CAF1 reduces the incidence of DSBs in TRC-prone RECQ5-deficient cells.

No abstract

In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription–replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.