The opposing activities of 53BP1 and BRCA1 influence pathway choice of DNA double-strand break repair. How BRCA1 counters the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2~ubiquitin. We demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitylation by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1 deficient cells. We show BRCA1-BARD1 function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin, optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning and the need for SMARCAD1 in Olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.Introduction.

The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and ␥ radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function-the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.Cells of all organisms have evolved an intricate series of DNA repair pathways to counteract the deleterious effects of all types of DNA damage. In Escherichia coli, nucleotide excision repair (NER) of UV damage requires the products of six genes. A complex of the UvrA and UvrB proteins binds to DNA and translocates to the site of the damage. The UvrC product then attaches to the complex, displacing UvrA, and the damaged DNA strand is nicked on both sides of the damaged site. The helicase activity of the UvrD product releases the oligonucleotide containing the damage, and DNA polymerase I and ligase complete the repair process (24). In eukaryotes, NER requires considerably more gene products, most of which are highly conserved (20). In Saccharomyces cerevisiae, the products of the RAD1, -2, -3, -4, -10, -14, and -25 genes are absolutely required for excision repair of UV damage, whereas there is only a partial requirement for RAD7, -16, and -23. There is evidence that the products of many of these genes form a multisubunit complex (e.g., see reference 53). In Schizosaccharomyces pombe, genes encoding highly homologous proteins have been identified (9,10,30,39), demonstrating the conservation of the classical NER pathway in this yeast.Null mutations in the S. cerevisiae NER genes RAD1, -2, -3, -10, and -14 result in a total deficiency in excision repair of UV-induced cyclobutane dimers and 6-4 photoproducts (28). Null mutants of the S. pombe homologs of RAD1 and RAD2 (rad16 and rad13, respectively), while showing many properties expected of excision repair-deficient mutants, are still able to excise UV-induced cyclobutane dimers and 6-4 photoproducts at a significant rate (7, 27). These results suggest that, in contrast to S. cerevisiae, there is a second pathway in S. pombe for removal of UV photoproducts.Most of the rad mutants of S. pombe are sensitive to both UV and ␥ irradiation. Some of these are involved in checkpoint control of the cell cycle to radiation (2, 3, 41). Others, which are particularly sensitive to ionizing radiation, are deficient in recombination repair (6,29,33,54). No mutant which is sensitive to ionizing but not to UV irradiation has yet been identified. The S. pombe rad18-X mutant ...

The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M r complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex.SUMO is a small ubiquitin-like protein that is covalently attached to target proteins. In yeasts and lower eukaryotes, SUMO is encoded by a single gene, while in higher eukaryotes there are three isoforms, SUMO-1, SUMO-2, and SUMO-3. The attachment of SUMO to target proteins is similar to the process of ubiquitination: SUMO is produced as a precursor protein which is processed to the mature form by SUMO proteases, revealing a diglycine motif. SUMO is subsequently activated by the formation of a thioester bond with a cysteine residue on the SUMO E1-like activator enzyme, a heterodimer known as SAE. SUMO is then passed to an E2-like SUMO conjugator, with which it also forms a thioester bond at a cysteine residue. SUMO ligases have been identified in several organisms. However, whereas E3 ligases are required for the attachment of ubiquitin to targets both in vitro and in vivo, the requirement for SUMO ligases for the attachment of SUMO to targets appears to be less stringent in vitro, and possibly also in vivo. This would be consistent with reports that several SUMO target proteins interact directly with the E2-like SUMO conjugator (e.g., see reference 4).Two classes of SUMO ligases have been identified. Proteins in the first category contain C3HC4-like RING domains, while proteins in the second category do not. Members of the first category include the Saccharomyces cerevisiae proteins Siz1 and Siz2 (16) and the mammalian PIAS family of proteins (20,32,38). Members of the second category include the RanBP2 and Pc proteins (18, 33). In S. cerevisiae (budding yeast), the SIZ1 and SIZ2 genes are not essential for viability, and null mutants do not show the severe cell and nuclear morphologies (16) that are observed with mutants that are defective in other components of the sumoylation system (17, 39). It remains...

The error-free and efficient repair of DNA double-stranded breaks (DSBs) is extremely important for cell survival. RNA has been implicated in the resolution of DNA damage but the mechanism remains poorly understood. Here, we show that miRNA biogenesis enzymes, Drosha and Dicer, control the recruitment of repair factors from multiple pathways to sites of damage. Depletion of Drosha significantly reduces DNA repair by both homologous recombination (HR) and non-homologous end joining (NHEJ). Drosha is required within minutes of break induction, suggesting a central and early role for RNA processing in DNA repair. Sequencing of DNA:RNA hybrids reveals RNA invasion around DNA break sites in a Drosha-dependent manner. Removal of the RNA component of these structures results in impaired repair. These results show how RNA can be a direct and critical mediator of DNA damage repair in human cells.

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono-and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.

The rad18 gene of Schizosaccharomyces pombe is an essential gene that is involved in several different DNA repair processes. Rad18 (Smc6) is a member of the structural maintenance of chromosomes (SMC) family and, together with its SMC partner Spr18 (Smc5), forms the core of a high-molecular-weight complex. We show here that both S. pombe and human Smc5 and -6 interact through their hinge domains and that four independent temperature-sensitive mutants of Rad18 (Smc6) are all mutated at the same glycine residue in the hinge region. This mutation abolishes the interactions between the hinge regions of Rad18 (Smc6) and Spr18 (Smc5), as does mutation of a conserved glycine in the hinge region of Spr18 (Smc5). We purified the Smc5-6 complex from S. pombe and identified four non-SMC components, Nse1, Nse2, Nse3, and Rad62. Nse3 is a novel protein which is related to the mammalian MAGE protein family, many members of which are specifically expressed in cancer tissue. In initial steps to understand the architecture of the complex, we identified two subcomplexes containing Rad18-Spr18-Nse2 and Nse1-Nse3-Rad62. The subcomplexes are probably bridged by a weaker interaction between Nse2 and Nse3.

A yeast gene MYO1 that contains regions of substantial sequence homology with the nematode muscle myosin gene (unc54) has been isolated and sequenced. Although the disruption of MYO1 is not lethal, it leads to aberrant nuclear migration and cytokinesis. The 200‐kd myosin heavy chain‐like protein, the product of MYO1, cross‐reacts with anti‐nematode myosin heavy chain IgG and is present in wild‐type strains but not in strains carrying the disrupted gene. Instead, a truncated polypeptide with a molecular mass of 120 kd can be detected in some myo1 mutants.

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.