Asian Pacific American Visual Artists in a Modern Seattle: 1960s to 1980s

The relationships, connections, and community networks of Asian American and Pacific Islander visual artists in the Seattle area reveal their responses to conditions wrought from a history of discrimination and economic inequality, overlaid with their own cultural heritage. From the 1960s to the 1980s, they formed bonds and shared concepts with kindred spirits, excelled in art achievements, and formed their own fellowships, creating a fabric of pan-Asian art. Moreover, non-Asian artists learned and benefited in crucial ways as they learned Asian art techniques and subject matter from API artists. After World War II, younger Asian American artists in Seattle rebuilt art-community ties. Community-focused art clubs nurtured and engaged artists of color, and less traditional venues augmented limited opportunities in galleries and museums for recognition. These artists found training opportunities in high schools and college-level art schools including the University of Washington and Cornish College of the Arts. The Wing Luke Asian Museum’s first Asian American invitational art exhibition, held in 1979 in its original space in the International District, featured 59 pan-Asian artists representing all art forms, selected by community-based curators. The success of this show encouraged the museum to continue to present fine arts by local API artists. From 1960s Jet Dreams to 21st Century Establishment Asian American and Pacific Islander artists, both native-born and those recently arrived from around the Pacific Rim, have come to expand the vocabulary and definitions of what makes the Seattle art world, some achieving renown and others still awaiting further recognition and celebration

The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50†

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF- 50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins.Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase

GEN1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes

AbstractProcessing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1

Disease Mapping and Spatial Regression with Count Data

In this paper we provide critical reviews of methods suggested for the analysis of aggregate count data in the context of disease mapping and spatial regression. We introduce a new method for picking prior distributions, and propose a number of refinements of previously-used models. We also consider ecological bias, mutual standardization, and choice of both spatial model and prior specification. We analyze male lip cancer incidence data collected in Scotland over the period 1975–1980, and outline a number of problems with previous analyses of these data. A number of recommendations are provided. In disease mapping studies, hierarchical models can provide robust estimation of area-level risk parameters, though care is required in the choice of covariate model, and it is important to assess the sensitivity of estimates to the spatial model chosen, and to the prior specifications on the variance parameters. Spatial ecological regression is a far more hazardous enterprise for two reasons. First, there is always the possibility of ecological bias, and this can only be alleviated via the inclusion of individual-level data. For the Scottish data we show that the previously used mean model has limited interpretation from an individual perspective. Second, when residual spatial dependence is modeled, and if the exposure has spatial structure, then estimates of exposure association parameters will change when compared with those obtained from the independence across space model, and the data alone cannot choose the form and extent of spatial correlation that is appropriate

Dissection of DNA double-strand-break repair using novel single-molecule forceps.

Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity

A conserved loop-wedge motif moderates reaction site search and recognition by FEN1

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognise opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3′‑terminus of a primer strand, which is recognised by breaking the terminal base pair to generate a substrate with a single nucleotide 3′‑flap. This recognition event allosterically signals hydrolytic removal of the 5′-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved ‘wedge’ residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated ‘loop–wedge’ mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognise irregular DNA structures. These new findings reveal how FEN1 precisely couples 3′-flap verification to function

Phosphate steering by Flap Endonuclease 1 promotes 5´-flap specificity and incision to prevent genome instability

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 50polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 50-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 50-flap specificity and catalysis, preventing genomic instability

Author response

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability. DOI: http://dx.doi.org/10.7554/eLife.21884.00