Mechanism of efficient double-strand break repair by a long non-coding RNA.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy

Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism

Oligomeric interface modulation causes misregulation of purine 5 nucleotidase in relapsed leukemia

Differential scanning fluorimetry of the C-terminally truncated cN-II variants in the presence or absence of 3 mM ATP. Truncated proteins are stabilized upon ATP binding, as described for full-length variants. Mean values and standard deviations from duplicates of two independent measurements are listed. (DOCX 14 kb

Conformationally constrained nucleoside phosphonic acids potent inhibitors of human mitochondrial and cytosolic 5 3 nucleotidases

Conformationally constrained nucleoside phosphonic acids – potent inhibitors of human mitochondrial and cytosolic 5′(3′)-deoxynucleotidases.</p