The SMC5/6 complex recruits the PAF1 complex to facilitate DNA double‐strand break repair in Arabidopsis

Abstract

DNA double‐strand breaks (DSBs) are one of the most toxic forms of DNA damage, which threatens genome stability. Homologous recombination is an error‐free DSB repair pathway, in which the evolutionarily conserved SMC5/6 complex (SMC5/6) plays essential roles. The PAF1 complex (PAF1C) is well known to regulate transcription. Here we show that SMC5/6 recruits PAF1C to facilitate DSB repair in plants. In a genetic screen for DNA damage response mutants (DDRMs), we found that the Arabidopsis ddrm4 mutant is hypersensitive to DSB‐inducing agents and is defective in homologous recombination. DDRM4 encodes PAF1, a core subunit of PAF1C. Further biochemical and genetic studies reveal that SMC5/6 recruits PAF1C to DSB sites, where PAF1C further recruits the E2 ubiquitin‐conjugating enzymes UBC1/2, which interact with the E3 ubiquitin ligases HUB1/2 to mediate the monoubiquitination of histone H2B at DSBs. These results implicate SMC5/6‐PAF1C‐UBC1/2‐HUB1/2 as a new axis for DSB repair through homologous recombination, revealing a new mechanism of SMC5/6 and uncovering a novel function of PAF1C.

Genome-wide evidence of the role of Paf1C in transcription elongation and histone H2B monoubiquitination inArabidopsis

“…Subsequently, Paf1C would promote post-translational modifications of histones such as monoubiquitination of H2B in nucleosomes downstream of the +1, thereby facilitating the passage of RNAPII thoughout the whole gene body where we found both Paf1C and H2Bub to frequently accumulate. The direct effect of Paf1C on local HUB1/2 activity is supported by several observations in this study, including the frequent cooccupancy of genes by Paf1C and RNAPII, with high levels of H2Bub in this context, the association of HUB1/2 with chromatin even in the absence of Paf1C, and the previously described physical interaction between the Paf1C subunit VIP5 and the Arabidopsis homologs of the Rad6 E2 ubiquitin-conjugating enzyme (Li et al, 2023). Nevertheless, the reduced H2Bub deposition in elf7 mutant plants could in some cases result from indirect effects of Paf1C deficiency, such as impaired RNAPII elongation.…”

“…In addition to flowering, and consistent with the pleiotropic effects of loss-offunction mutants in several subunits, plant Paf1C has been associated genetically with a variety of largely unrelated physiological processes in Arabidopsis, including seed dormancy (Liu et al, 2011), shoot apical meristem activity (Fal et al, 2017;Fal et al, 2019;Li et al, 2022), thermomorphogenesis (Zhao et al, 2023), and the response to salt stress (Zhang et al, 2022) and DNA damaging agents (Li et al, 2023).…”

“…In line with the role of Paf1C in animals and fugi in promoting H2Bub accumulation, mutations in Arabidopsis VIP2, VIP4, VIP5, and VIP6 subunits cause decreased H2Bub accumulation at FLC and its paralog genes (Cao et al, 2015). More recently, the dependence of H2Bub on Paf1C has been linked to the DNA damage response (Li et al, 2023). In Arabidopsis , as in yeast and animals, H2Bub accumulates across the gene bodies of actively transcribed genes (Roudier et al, 2011; Bourbousse et al, 2012; Nassrallah et al, 2018).…”

The plant POLYMERASE-ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome wide

“…In addition to flowering, and consistent with the pleiotropic effects of loss-of-function mutants in several subunits, plant Paf1C has been associated genetically with a variety of largely unrelated physiological processes in Arabidopsis , including seed dormancy ( Liu et al 2011 ), shoot apical meristem activity ( Fal et al 2017 ; Fal et al 2019 ; Li et al 2022 ), thermomorphogenesis ( Zhao et al 2023 ), and the response to salt stress ( Zhang et al 2022 ) and DNA damaging agents ( Li et al 2023 ).…”

“…In line with the role of Paf1C in animals and fungi in promoting H2Bub accumulation, mutations in Arabidopsis VIP2 , VIP4 , VIP5 , and VIP6 subunits cause decreased H2Bub accumulation at FLC and its paralog genes ( Cao et al 2015 ). More recently, the dependence of H2Bub on Paf1C has been linked to the DNA damage response ( Li et al 2023 ). In Arabidopsis , as in yeast and animals, H2Bub accumulates across the gene bodies of actively transcribed genes ( Roudier et al 2011 ; Bourbousse et al 2012 ; Nassrallah et al 2018 ).…”