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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., (© 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Homologous recombination (HR) is a key DNA damage repair pathway that is tightly adjusted to the state of a cell. A central regulator of homologous recombination is the conserved helicase‐containing Bloom syndrome complex, renowned for its crucial role in maintaining genome integrity. Here, we show that in Arabidopsis thaliana, Bloom complex activity is controlled by selective autophagy. We find that the recently identified DNA damage regulator KNO1 facilitates K63‐linked ubiquitination of RMI1, a structural component of the complex, thereby triggering RMI1 autophagic degradation and resulting in increased homologous recombination. Conversely, reduced autophagic activity makes plants hypersensitive to DNA damage. KNO1 itself is also controlled at the level of proteolysis, in this case mediated by the ubiquitin–proteasome system, becoming stabilized upon DNA damage via two redundantly acting deubiquitinases, UBP12 and UBP13. These findings uncover a regulatory cascade of selective and interconnected protein degradation steps resulting in a fine‐tuned HR response upon DNA damage. Synopsis: KNOTEN1 (KNO1) is a recently identified RBR1 transcriptional target involved in the DNA damage response in Arabidopsis thaliana. Functional characterization of KNO1 reveals it as a regulator of homologous recombination repair via damage‐dependent degradation of the core Bloom syndrome complex component RMI1.KNO1 mediates K63‐linked ubiquitination of RMI1 to trigger its degradation via autophagyAutophagic degradation of RMI1 facilitates homologous recombination after DNA damage.In the absence of DNA damage, KNO1 is constitutively degraded via the ubiquitin–proteasome pathway.KNO1 becomes stabilized after DNA damage via de‐ubiquitination by UBP12 and UBP13.Reduced autophagy sensitizes plants to DNA damage. [ABSTRACT FROM AUTHOR]

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress. [ABSTRACT FROM AUTHOR]

Upon DNA damage, cyclin-dependent kinases ( CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair ( HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs ( CDKB1s) and the class of B1-type cyclins ( CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 ( RAD51), a core mediator of HR, is a substrate of CDKB1- CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 ( SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1- CYCB1 complexes are specifically activated to mediate HR. [ABSTRACT FROM AUTHOR]