DNA repair by homologous recombination (HR)1 is highly suppressed in G1 cells2,3 to ensure that mitotic recombination occurs solely between sister chromatids4. Although many HR factors are cell cycle-regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 in order to constrain BRCA2 function to the S/G2 phases. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein5, in complex with CUL3-RBX16. PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA end resection is sufficient to induce HR in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR/Cas9-based gene targeting assay. We conclude that the mechanism prohibiting HR in G1 minimally consists of the suppression of DNA end resection coupled to a multi-step block to BRCA2 recruitment to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce HR in G1 cells with defined factors could spur the development of gene targeting applications in non-dividing cells.

DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR)1–3. However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ)4. We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells4, 5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

No abstract

Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP1 1,2 , mediators of the homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair pathways, respectively 3 . While NHEJ relies on 53BP1 binding directly to ubiquitinated Lysine 15 on H2A-type histones (H2AK15ub) 4,5 -an RNF168dependent modification 6 -how RNF168 promotes BRCA1 recruitment and function remains unclear. Here, we identify a tandem BRCT domain-associated ubiquitin-dependent recruitment motif (BUDR) in BARD1 -BRCA1's obligate partner proteinthat by engaging H2AK15ub, recruits BRCA1 to DSBs. BARD1 BUDR disruption compromises HR and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 Lys20 (H4K20me0) by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes HR activities of the BRCA1-BARD1 complex. Finally, genetic epistasis experiments confirm that the need for BARD1-chromatin binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA damagedependent and post-replication histone PTM states, BRCA1-BARD1 complexes coordinate 53BP1 pathway antagonization with HR promotion, establishing a simple paradigm for the governance of DSB repair pathway choice.

Given the predominance of outcrossing by angiosperms, large costs must often overwhelm the genetic benefit of selfing derived from contributing two haploid genomes to each off-spring rather than one. In addition to the well-studied genetic cost of inbreeding depression, selfing imposes a mating cost whenever self-pollination reduces opportunities for pollen export. Because self-pollination is a heterogeneous process, pollen discounting and its evolutionary consequences vary with pollination conditions. In this article we model self-pollination as comprising discounting and nondiscounting components, and we consider the consequences of this heterogeneity for outcross siring success. Aided by this depiction of pollination, we then compare previous theoretical representations of pollen discounting and consider their relative virtues. Finally, we consider conditions that would allow a population to be invaded by a variant with different pollination characteristics. This analysis exposes the pollination conditions implicit in standard results of mating system theory. It also identifies associations between four possible changes in pollination expected in different reproductive environments, including the incidence of positive or negative correlations between self-pollination and pollen export. These results emphasize the benefits of expanding the theory of plant reproduction to recognize explicitly when and how pollination mechanisms affect mating outcomes.

The owl monkey, as do other mammals, has X-and Y-cells in its lateral geniculate nucleus. X-cells are found in the parvocellular laminae; Y-cells, in the magnocellular laminae.

SummaryDNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a “mechanism of last resort” employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function.