Abstract
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.
Highlights
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
We discovered that the conjugation of Proliferating cell nuclear antigen (PCNA) to SUMO2, but not small ubiquitin-like modifier 1 (SUMO1), is induced by transcription and is unique to the replication fork associated with the transcribed region of the chromatin
Through this study, we discovered the specific association between K164 SUMO2-conjugated human PCNA and transcribed chromatin (Fig. 1)
Summary
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. We further determined that the RNAPII-bound DNA helicase RECQ5, which suppresses transcription-associated DSBs and acts as a tumor suppressor[14,15,16,17,18,19,20], interacts with PCNA via a newly identified PCNA-interacting protein (PIP) motif to induce SUMO2 conjugation of PCNA. Increasing SUMO2-PCNA levels in cells enhances deposition of CAF1-dependent histone H3.1 and establishment of repressive histone marks, which coincide with the destabilization of RNAPII from the chromatin, especially at CFSs, and enhanced replication fork progression. Through these mechanisms, overexpression of SUMO2-PCNA or CAF1 reduces incidents of DSBs in TRC-prone RECQ5-deficient cells. Our observations provide mechanistic insight into how SUMO2-PCNA restricts transcription via chromatin remodeling during DNA replication to minimize genomic instability that may arise from catastrophic encounters between the replication and transcription machinery
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