The retinoblastoma homolog RBR1 mediates localization of the repair protein RAD51 to DNA lesions in Arabidopsis

Abstract

The retinoblastoma protein (Rb), which typically functions as a transcriptional repressor of E2F‐regulated genes, represents a major control hub of the cell cycle. Here, we show that loss of the Arabidopsis Rb homolog RETINOBLASTOMA‐RELATED 1 (RBR1) leads to cell death, especially upon exposure to genotoxic drugs such as the environmental toxin aluminum. While cell death can be suppressed by reduced cell‐proliferation rates, rbr1 mutant cells exhibit elevated levels of DNA lesions, indicating a direct role of RBR1 in the DNA‐damage response (DDR). Consistent with its role as a transcriptional repressor, we find that RBR1 directly binds to and represses key DDR genes such as RADIATION SENSITIVE 51 (RAD51), leaving it unclear why rbr1 mutants are hypersensitive to DNA damage. However, we find that RBR1 is also required for RAD51 localization to DNA lesions. We further show that RBR1 is itself targeted to DNA break sites in a CDKB1 activity‐dependent manner and partially co‐localizes with RAD51 at damage sites. Taken together, these results implicate RBR1 in the assembly of DNA‐bound repair complexes, in addition to its canonical function as a transcriptional regulator.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

“…AtBRCA1 and RBR are independently recruited to these specific damage foci, they interact and our genetic study show that they partially act together to maintain genome integrity and to prevent cell death. The accompanying paper of Biedermann et al () shows that RBR is required to localise RAD51 and that RBR and RAD51 co‐localise in these large foci, corroborating a non‐transcriptional role for RBR in the maintenance of plant genome integrity. We further show that RBR and E2FA also have transcriptional DNA damage response roles that act in parallel to the well‐established SOG1 pathway.…”

Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress

“…The particularly high sensitivity of h2ax mutant plants to the DNA crosslinking reagent MMC relative to other forms of DNA damage mirrors phenotypic analysis of mutants in homologous recombination (HR), including the main recombinase RAD51 and its paralogues (Bleuyard et al ., ). Potential function for γ‐H2AX in promotion of HR activity in plants is supported by the recent finding that RAD51 colocalizes with γ‐H2AX in response to DNA damage (Biedermann et al ., ). Taken together with the altered pattern in end‐joining products previously identified in H2AX‐deficient lines (Qi et al ., ) and the differential sensitivity of h2ax‐2 mutant seeds to ageing observed in the present study, these results indicate that in plants H2AXA influences multiple pathways for the repair of DSBs and is not redundant in function to H2AXB.…”

“…Interaction of MEDIATOR OF THE DNA DAMAGE CHECKPOINT 1 (MDC1) with phosphorylated H2AX potentiates the DNA damage response in mammals (Lukas et al ., ), while downstream factors recruited to repair foci remain largely unknown in plants. However, fluorescence microscopy identified partial co‐localization of damaged‐induced γ‐H2AX foci in Arabidopsis with E2FA, a transcription factor involved in regulating genes required for DNA replication and cell cycle control (Lang et al ., ), and recent studies localized RAD51, BRCA1 and RBR to γ‐H2AX foci (Biedermann et al ., ; Horvath et al ., ). Here we showed that H2AX phosphorylation during 100 Gy (100 min) genotoxin exposure was entirely dependent on ATM, with no significant phosphorylation observed in the atm‐3 mutant.…”

Negative regulator of E2F transcription factors links cell cycle checkpoint and DNA damage repair

“…Recently, two independent research groups demonstrated that RBR1 directly regulates DNA repair (55,56). The rbr1 mutant is hypersensitive to DNA-damaging agents.…”