SUMO2 conjugation of PCNA facilitates chromatin remodeling to resolve transcription-replication conflicts

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.

PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

“…Previously, PCNA SUMOylation has been implicated in regulating chromatin structure at transcribed loci (Li et al, 2018). Our work suggests that this does not affect fork protection, as we show that loss of PCNA ubiquitination, but not of SUMOylation, results in nascent strand degradation.…”

“…The K164 residue of PCNA is subjected not only to ubiquitination, but also to SUMOylation (Li et al, 2018;Moldovan et al, 2012). Depletion of the ubiquitin ligase RAD18 recapitulated the PCNA-K164R phenotype, as it resulted in DNA2-mediated nascent tract degradation.…”

Transcription-mediated replication hindrance: a major driver of genome instability

“…On the other hand, yeast and human cells lacking the FACT complex showed transcription-associated genetic instability and fork progression impairments, implying a key role of this histone chaperone to prevent T-R conflicts (Herrera-Moyano et al 2014). In agreement, the histone chaperones FACT and CAF1 are specifically recruited to transcribing chromatin to promote fork progression (Li et al 2018). Other chromatin factors could also inherently protect the cell from T-R conflicts by regulating the coordination between DNA replication and transcription, and, indeed, depletion of histone H1, a key heterochromatin component, causes replication stress and DNA damage linked to T-R conflicts in Drosophila and human cells (Bayona-Feliu et al 2017;Almeida et al 2018).…”

RECQ5: A Mysterious Helicase at the Interface of DNA Replication and Transcription

“…RECQ5 structure can be divided into two parts: the conserved N-terminal region including the RecA-like helicase domain (residues 1-364) and Zn 2+ -binding domain (residues 365-437) [16,17], and a unique C-terminal region (residues 438-991) harboring at least five specific protein interaction domains: RAD51-binding domain, internal RNAPII-interacting (IRI) domain that binds to hypophosphorylated form of RNAPII (RNAPIIa), Set2-Rpb1-interacting (SRI) domain that binds to the hyperphosphorylated (elongating) form of RNAPII (RNAPIIo), RNA polymerase I (RNAPI)-binding domain and PCNA-interacting protein (PIP) motifs [18][19][20][21][22][23][24][25][26][27] (Figure 1). The helicase domain consists of two RecA-like domains, D1 and D2, each containing a central 6-or 7-stranded parallel β-sheet flanked on either side by α-helices [17].…”