The ability to sense and respond to DNA damage is critical to maintenance of genomic stability and the prevention of cancer. In this study, we employed a genetic screen to identify a gene, NBA1 (new component of the BRCA1 A complex), that is required for resistance to ionizing radiation. The NBA1 protein localizes to sites of DNA damage and is required for G2/M checkpoint control. Proteomic analysis revealed that NBA1 is a component of the BRCA1 A complex, which also contains Brca1/Bard1, Abra1, RAP80, BRCC36, and BRE. NBA1 is required to maintain BRE and Abra1 abundance and for the recruitment of BRCA1 to sites of DNA damage. In depth bioinformatics analysis revealed that the BRCA1 A complex bears striking similarities to the 19S proteasome complex. Furthermore, we show that four members of the BRCA1-A complex possess a polyubiquitin chainbinding capability, thus forming a complex that might facilitate the deubiquitinating activity of the deubiquitination enzyme BRCC36 or the E3 ligase activity of the BRCA1/BARD1 ligase. These findings provide a new perspective from which to view the BRCA1 A complex.[Keywords: Abraxas; BRCA1 A complex; MPN domain; proteasome; VWA domain] Supplemental material is available at http://www.genesdev.org.

Background: Ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) conjugation occurs at DNA double-strand breaks (DSBs). Results: Rap80, a component of the BRCA1-A complex, binds to both SUMO and Ub conjugates. Conclusion: Rap80 binding to both SUMO and Ub conjugates is required for proper cellular response to DSBs. Significance: This work provides insights into how Rap80 and BRCA1 are recruited to DSBs to maintain genome stability.

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A–F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

Dysregulated inflammation is a complicated pathological process involved in various diseases, and the treatment of inflammation-linked disorders currently represents an enormous global burden. Extracellular vesicles (EVs) are nanosized, lipid membrane-enclosed vesicles secreted by virtually all types of cells, which act as an important intercellular communicative medium. Considering their capacity to transfer bioactive substances, both unmodified and engineered EVs are increasingly being explored as potential therapeutic agents or therapeutic vehicles. Moreover, as the nature's own delivery tool, EVs possess many desirable advantages, such as stability, biocompatibility, low immunogenicity, low toxicity, and biological barrier permeability. The application of EV-based therapy to combat inflammation, though still in an early stage of development, has profound transformative potential. In this review, we highlight the recent progress in EV engineering for inflammation targeting and modulation, summarize their preclinical applications in the treatment of inflammatory disorders, and present our views on the anti-inflammatory applications of EV-based nanotherapeutics.

Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotidebinding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys 114 at the nucleotidebinding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to lossof-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G 1 /S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotidebinding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATPbinding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G 1 /S transition.Cell cycle regulation is a complicated but highly coordinated process. It has a conserved mechanism among eukaryotes from yeast to human. The primary control of the eukaryotic cell cycle is provided by a family of cyclin-dependent kinases (CDKs) 1 and their associated cyclins, which regulate kinase activity. In unicellular yeast cells, a single prototype CDK gene, CDC28 in the budding yeast Saccharomyces cerevisiae or cdc2 ϩ in the fission yeast Schizosaccharomyces pombe functions at different cell cycle stages. Different cyclins activate the same kinase as different points in the cycle. It is now known that the central control of cell cycle progression by CDK complexes is regulated positively and negatively to monitor each step of the progression (1, 2). This regulatory control, associated with checkpoints, orchestrates various types of cell cycle ge...