BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system’s specificity. Recently a “double-nicking” strategy using catalytic mutant Cas9D10A nickase has been developed to minimise off-target effects. Here, we describe a Cas9D10A-based screening approach that combines an All-in-One Cas9D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.

Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn 2؉ . These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5 splice site and the branch point during the first catalytic reaction.Precursor mRNAs (pre-mRNAs) excise their introns via two steps of a transesterification reaction. The reaction takes place on a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, U6, and numerous protein factors. The spliceosome is a highly dynamic structure, formed by stepwise binding to the pre-mRNA of snRNAs in the form of small nuclear ribonucleoprotein complexes (snRNPs) (for a review, see references 3, 29, and 35-37). Following the binding of all snRNAs, the spliceosome undergoes a major structural change, leading to the release of U1 and U4 and the formation of the active spliceosome that is able to carry out the catalytic reaction.Spliceosome activation also requires a large protein complex, the Prp19-associated complex (nineteen complex, or NTC), which is added to the spliceosome after the release of U1 and U4 to stabilize the association of U5 and U6 with the spliceosome (5). The NTC plays an important role in promoting or stabilizing high-specificity interactions between U6 and the 5Ј splice site and between U5 and the exon sequence at the splice junctions after U1 and U4 have dissociated (4, 5). Eight components of the NTC have been identified, including Prp19,

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Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter ␥-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD CAT . The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD CAT exhibited substrate specificity to L-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD CAT for GABAergic therapies or neuroprotection.

bYju2 is an essential splicing factor required for the first catalytic step after the action of Prp2. We dissected the structure of Yju2 and found that the amino (Yju2-N) and carboxyl (Yju2-C) halves of the protein can be separated and reconstituted for Yju2 function both in vivo and in vitro. Yju2-N has a weak affinity for the spliceosome but functions in promoting the first reaction, with the second reaction being severely impeded. The association of Yju2-N with the spliceosome is stabilized by the presence of Yju2-C at both the precatalytic and postcatalytic stages. Strikingly, Yju2-N supported a low level of the second reaction even in the absence of Prp16. Prp16 is known to mediate destabilization of Yju2 and Cwc25 after the first reaction to allow progression of the second reaction. We propose that in the absence of the C domain, Yju2-N is not stably associated with the spliceosome after lariat formation, and thus bypasses the need for Prp16. We also showed, by UV cross-linking, that Yju2 directly contacts U2 snRNA primarily in the helix II region both pre-and postcatalytically and in the branch-binding region only at the precatalytic stage, suggesting a possible role for Yju2 in positioning the branch point during the first reaction. Introns are removed from precursor mRNA via two steps of transesterification reactions that form lariat intermediates and products. The reactions are catalyzed by a large ribonucleoprotein complex, the spliceosome, which consists of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, in the form of small nuclear ribonucleoprotein particles (snRNPs), and numerous protein factors (for reviews, see references 1 to 4). The spliceosome is a highly dynamic structure, assembled by sequential binding of the snRNAs in the order U1, U2, and then U4/U6.U5 as a preformed tri-snRNP. U1 and U2 play roles in mediating the recognition of the 5= splice site and the branch site, respectively, through base pairing between the snRNAs and the intron sequences. Following binding of the tri-snRNP, the spliceosome undergoes a major structural rearrangement, releasing U1 and U4 and forming new base pairs between U6 and the 5= splice site and between U2 and U6. A protein complex associated with Prp19, called NTC (nineteen complex), is added to the spliceosome to stabilize the association of U5 and U6 with the spliceosome (5-7), and this proceeds with catalytic activation of the spliceosome. The NTC remains associated with the spliceosome until completion of the reaction.The spliceosome undergoes extensive remodeling throughout the splicing pathway (3, 8). Eight DEXD/H-box ATPases are required to mediate structural changes of the spliceosome during the splicing process (9). Prp2 and Prp16 are required for the first and the second catalytic step, respectively. Several other protein factors are required to promote the catalytic reactions following their actions. Prp2 has recently been shown to function in destabilizing the U2 components SF3a and SF3b (8,10,11). SF3b is known to interact with the br...

The regulatory domain (PA3346RS), comprising the receiver and stalk domains, of the response regulator PA3346 requires phosphorylation for activation with magnesium ions as cofactors in order to modulate the downstream protein phosphatase activity for the regulation of swarming motility in Pseudomonas aeruginosa PAO1. Fusion‐tagged recombinant PA3346RS of total molecular mass 25.3 kDa has been overexpressed in Escherichia coli, purified using Ni2+–NTA and Q‐Sepharose ion‐exchange columns and crystallized using the hanging‐drop vapour‐diffusion method. X‐ray diffraction data were collected from PA3346RS crystals to 2.0 Å resolution. The crystal belonged to space group P41 or P43, with unit‐cell parameters a = 82.38, c = 73.34 Å. Preliminary analysis indicated the presence of a dimer of PA3346RS in the asymmetric unit, with a solvent content of 48.6%.